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    Structured Review

    Thermo Fisher a549 cells
    Overexpression of circPTK2 enhances TIF1γ expression and inhibits TGF-β-induced EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in <t>A549</t> and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P
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    1) Product Images from "Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer"

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0889-7

    Overexpression of circPTK2 enhances TIF1γ expression and inhibits TGF-β-induced EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in A549 and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P
    Figure Legend Snippet: Overexpression of circPTK2 enhances TIF1γ expression and inhibits TGF-β-induced EMT and invasion of NSCLC cells in vitro. a TIF1γ mRNA and protein levels in A549 and H226 cells transiently overexpressing circPTK2. Relative TIF1γ expression was determined with normalization against β-actin. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing circPTK2 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 0.5 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. c A549 and H226 cells transiently overexpressing circPTK2 were serum-starved for 24 h and then treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Snail and N-cadherin protein levels were determined by western blot. β-actin was used as internal control. d , e A549 and H226 cells transiently overexpressing circPTK2 were treated as above and subjected to the transwell migration and invasion assays. Migrated and invasive cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. * P

    Techniques Used: Over Expression, Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Migration, Staining

    CircPTK2 overexpression attenuates NSCLC cell metastasis in vivo, and circPTK2 levels were lower in metastatic NSCLC tissues than non-metastatic counterparts. a CircPTK2 expression in A549 cells stably overexpressing circPTK2. A549 stable cell line overexpressing circPTK2 was generated as described in Methods. pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper ) was used to stably overexpress circPTK2. The empty vector was served as negative control. CircPTK2 expression was determined by qRT-PCR ( bottom left ). CircPTK2 expression in circPTK2-overexpressed A549 cells was determined using northern blots. RNase R was used to digest linear RNA ( bottom right ). b Schematic flowchart of the in vivo metastasis experiments with A549 cells stably transfected with pLCDH-circPTK2 or vector (i.v.) and TGF-β1 (i.p.) injected into BALB/c nude mice ( n = 6 mice per group in circPTK2 + TGF-β1 and vector + TGF-β1). c Representative images showing metastatic nodules established in lung taken from the mice injected with circPTK2-overexpressed A549 cells or vector control cells ( upper ). Scale bar, 4 mm. Haematoxylin and eosin (H E) staining was performed for histological confirmation of metastasizing tumor cells in lung ( bottom ). Scale bar, 100 μm. d Gross view of metastatic nodules developed in liver ( upper left ) and dot plots showing the number of metastatic nodules in liver ( upper right , n = 6 mice per group). Scale bar, 4 mm. Microscopic images of H E staining for liver metastases ( bottom left ) and the distribution of the number of metastases in per section of liver ( bottom right , n = 6 mice per group). Scale bar, 100 μm. Yellow and green arrowheads indicate metastatic nodules and micrometastases. e Representative images indicating metastatic nodules developed in pericardium ( upper , n = 6 mice per group) and H E staining of heart ( bottom ). Scale bar, 1 mm or 100 μm. f , g CircPTK2 and TIF1γ mRNA expression levels in human lung epithelial and NSCLC cells. β-actin was used as internal control. Each qRT-PCR analysis was performed in triplicate. h , i qRT-PCR analysis of circPTK2 and TIF1γ mRNA levels in 73 human NSCLC tissues and paired noncancerous lung tissues. Mean values are indicted by solid bars, and values are expressed as mean ± SEM. T, NSCLC tissues; N, paired noncancerous lung tissues. j , k Relative expression of circPTK2 and TIF1γ mRNA in 73 paired NSCLC tissues. Y -axis represents the log 10 transformed fold change of T/N expression ratios of circPTK2 and TIF1γ mRNA. The number of each specimen is shown below x -axis. l Correlation between circPTK2 level and TIF1γ mRNA expression in 73 paired NSCLC tissues. X and y axes represent the T/N expression ratios of circPTK2 and TIF1γ mRNA, respectively. m Relative expression (T/N) of circPTK2 in metastatic ( n = 41) and non-metastatic ( n = 32) NSCLC tissues. Metastatic tissues were from NSCLC patients with lymph node metastasis or distant metastasis and non-metastatic tissues were from NSCLC patients without any metastasis, respectively. * P
    Figure Legend Snippet: CircPTK2 overexpression attenuates NSCLC cell metastasis in vivo, and circPTK2 levels were lower in metastatic NSCLC tissues than non-metastatic counterparts. a CircPTK2 expression in A549 cells stably overexpressing circPTK2. A549 stable cell line overexpressing circPTK2 was generated as described in Methods. pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper ) was used to stably overexpress circPTK2. The empty vector was served as negative control. CircPTK2 expression was determined by qRT-PCR ( bottom left ). CircPTK2 expression in circPTK2-overexpressed A549 cells was determined using northern blots. RNase R was used to digest linear RNA ( bottom right ). b Schematic flowchart of the in vivo metastasis experiments with A549 cells stably transfected with pLCDH-circPTK2 or vector (i.v.) and TGF-β1 (i.p.) injected into BALB/c nude mice ( n = 6 mice per group in circPTK2 + TGF-β1 and vector + TGF-β1). c Representative images showing metastatic nodules established in lung taken from the mice injected with circPTK2-overexpressed A549 cells or vector control cells ( upper ). Scale bar, 4 mm. Haematoxylin and eosin (H E) staining was performed for histological confirmation of metastasizing tumor cells in lung ( bottom ). Scale bar, 100 μm. d Gross view of metastatic nodules developed in liver ( upper left ) and dot plots showing the number of metastatic nodules in liver ( upper right , n = 6 mice per group). Scale bar, 4 mm. Microscopic images of H E staining for liver metastases ( bottom left ) and the distribution of the number of metastases in per section of liver ( bottom right , n = 6 mice per group). Scale bar, 100 μm. Yellow and green arrowheads indicate metastatic nodules and micrometastases. e Representative images indicating metastatic nodules developed in pericardium ( upper , n = 6 mice per group) and H E staining of heart ( bottom ). Scale bar, 1 mm or 100 μm. f , g CircPTK2 and TIF1γ mRNA expression levels in human lung epithelial and NSCLC cells. β-actin was used as internal control. Each qRT-PCR analysis was performed in triplicate. h , i qRT-PCR analysis of circPTK2 and TIF1γ mRNA levels in 73 human NSCLC tissues and paired noncancerous lung tissues. Mean values are indicted by solid bars, and values are expressed as mean ± SEM. T, NSCLC tissues; N, paired noncancerous lung tissues. j , k Relative expression of circPTK2 and TIF1γ mRNA in 73 paired NSCLC tissues. Y -axis represents the log 10 transformed fold change of T/N expression ratios of circPTK2 and TIF1γ mRNA. The number of each specimen is shown below x -axis. l Correlation between circPTK2 level and TIF1γ mRNA expression in 73 paired NSCLC tissues. X and y axes represent the T/N expression ratios of circPTK2 and TIF1γ mRNA, respectively. m Relative expression (T/N) of circPTK2 in metastatic ( n = 41) and non-metastatic ( n = 32) NSCLC tissues. Metastatic tissues were from NSCLC patients with lymph node metastasis or distant metastasis and non-metastatic tissues were from NSCLC patients without any metastasis, respectively. * P

    Techniques Used: Over Expression, In Vivo, Expressing, Stable Transfection, Generated, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Northern Blot, Transfection, Injection, Mouse Assay, Staining, Transformation Assay

    TIF1γ and circPTK2 are down-regulated during TGF-β-induced EMT in NSCLC cells. a A549 cells underwent epithelial-mesenchymal transition (EMT) after TGF-β1 (5 ng/ml) treatment for 24 h. Cell morphology was observed and photographed with a phase-contrast microscope ( upper ). Scale bar, 50 μm. The expression of EMT-related makers including E-cadherin, N-cadherin and Vimentin ( bottom left ), and TIF1γ protein ( bottom right ) were examined by western blot. β-actin was used as internal control. Densitometry values for each protein were normalized to β-actin and shown below the corresponding bands. b RNA from epithelial and mesenchymal A549 cells were subjected to Arraystar Human circRNA Array analysis as described in Methods. Hierarchical cluster analysis (heat map) of microarray data was used to show the significant expression of circRNAs when comparing mesenchymal cells with epithelial cells ( left ). Red and green denoted high and low expression, respectively. Each column represents a test sample and each row represents a circRNA. Each group (treated with TGF-β1 for 0 h or 24 h) was analyzed in triplicate. In a zoomed-in view of partial ( right ), the expression of circPTK2 (hsa_circRNA_104703) was indicated as an arrow. c The sketch of genomic locus of circPTK2 in PTK2 gene. The expression of circPTK2 (circBase ID: hsa_circ_0008305) was validated by RT-PCR followed by sanger sequencing. Red arrows represent divergent primers, which are used to amplify the genome region of circPTK2 containing the back-splice junction site (JCT). d In A549 or H226 cells, divergent primers amplify circPTK2 JCT in cDNA but not in genomic DNA (gDNA), convergent primers amplify both circPTK2 JCT and linear PTK2 Exon 9. GAPDH was used as linear control. Red and black arrows represent divergent and convergent primers, respectively. Divergent primers spanning circPTK2 JCT yield a product of 110 bp, while the convergent primers amplifying PTK2 exon 9 yield a product of 141 bp. e Endogenous circPTK2 expression in A549 cells was validated by northern blots. RNase R was used to digest linear RNA. f Representative image of RNA fluorescence in situ hybridization for endogenous circPTK2 in A549 cells. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. g qRT-PCR analysis of circPTK2 expression in A549 and H226 cells treated with TGF-β1 for 24 h. Relative circPTK2 expression was determined with normalization against β-actin. h, i qRT-PCR analysis of miR-429/miR-200b-3p expression levels in A549 and H226 cells treated with TGF-β1 for 24 h. U6 was used as internal control. * P
    Figure Legend Snippet: TIF1γ and circPTK2 are down-regulated during TGF-β-induced EMT in NSCLC cells. a A549 cells underwent epithelial-mesenchymal transition (EMT) after TGF-β1 (5 ng/ml) treatment for 24 h. Cell morphology was observed and photographed with a phase-contrast microscope ( upper ). Scale bar, 50 μm. The expression of EMT-related makers including E-cadherin, N-cadherin and Vimentin ( bottom left ), and TIF1γ protein ( bottom right ) were examined by western blot. β-actin was used as internal control. Densitometry values for each protein were normalized to β-actin and shown below the corresponding bands. b RNA from epithelial and mesenchymal A549 cells were subjected to Arraystar Human circRNA Array analysis as described in Methods. Hierarchical cluster analysis (heat map) of microarray data was used to show the significant expression of circRNAs when comparing mesenchymal cells with epithelial cells ( left ). Red and green denoted high and low expression, respectively. Each column represents a test sample and each row represents a circRNA. Each group (treated with TGF-β1 for 0 h or 24 h) was analyzed in triplicate. In a zoomed-in view of partial ( right ), the expression of circPTK2 (hsa_circRNA_104703) was indicated as an arrow. c The sketch of genomic locus of circPTK2 in PTK2 gene. The expression of circPTK2 (circBase ID: hsa_circ_0008305) was validated by RT-PCR followed by sanger sequencing. Red arrows represent divergent primers, which are used to amplify the genome region of circPTK2 containing the back-splice junction site (JCT). d In A549 or H226 cells, divergent primers amplify circPTK2 JCT in cDNA but not in genomic DNA (gDNA), convergent primers amplify both circPTK2 JCT and linear PTK2 Exon 9. GAPDH was used as linear control. Red and black arrows represent divergent and convergent primers, respectively. Divergent primers spanning circPTK2 JCT yield a product of 110 bp, while the convergent primers amplifying PTK2 exon 9 yield a product of 141 bp. e Endogenous circPTK2 expression in A549 cells was validated by northern blots. RNase R was used to digest linear RNA. f Representative image of RNA fluorescence in situ hybridization for endogenous circPTK2 in A549 cells. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. g qRT-PCR analysis of circPTK2 expression in A549 and H226 cells treated with TGF-β1 for 24 h. Relative circPTK2 expression was determined with normalization against β-actin. h, i qRT-PCR analysis of miR-429/miR-200b-3p expression levels in A549 and H226 cells treated with TGF-β1 for 24 h. U6 was used as internal control. * P

    Techniques Used: Microscopy, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Sequencing, Northern Blot, Fluorescence, In Situ Hybridization, Quantitative RT-PCR

    CircPTK2 binds directly to miR-429/miR-200b-3p in NSCLC cells. a Schematic description for the subcloning of the predicted miR-429/miR-200b-3p binding sites of circPTK2 exon11 in psiCHECK-2 luciferase vector. Predicted duplex formation between miR-429/miR-200b-3p and the wild-type/mutant of miR-429/miR-200b-3p binding sites of circPTK2 exon11 was shown. The entire subcloning sequences were listed in Additional file 4 : Table S2. b , c Relative luciferase activity of the wild-type/mutant circPTK2 exon11 reporter gene in A549 and H226 cells transfected with miR-429/miR-200b-3p or negative control (miR-NC). Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity was determined after normalizing against the firefly luciferase activity. d According to the flowchart outlining the experimental procedures ( left ), anti-AGO2 RIP was conducted in A549 cells transiently overexpressing miR-429/miR-200b-3p or miR-NC, and followed by RT-PCR and gel-staining analyses to detect circPTK2 enrichment ( right ). The 10% input was obtained as positive control before immunoprecipitation, and subjected to RT-PCR to confirm the presence of circPTK2. Anti-IgG antibody was used as a negative control. Red arrows represent divergent primers spanning the back-splice junction site of circPTK2. e According to the RNA pull-down flowchart ( left ), whole-cell lysates from A549 cells were incubated with biotinylated probes against circPTK2; after pull-down, endogenous circPTK2 ( middle ) and miR-429/miR-200b-3p ( right ) enrichments were detected by qRT-PCR. Results were presented as the percentage of pull-down to input. PD, pull-down. f Co-localization between circPTK2 (red) and miR-429/miR-200b-3p (green) was observed (arrowheads) by fluorescence in situ hybridization in A549 cells. Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. g CircPTK2 expression in A549 and H226 cells transiently overexpressing circPTK2. Cells were transiently transfected with pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper panel ) for 48 h or 72 h and then subjected to qRT-PCR analysis ( bottom panel ). The empty vector was served as negative control. In the upper panel, the subcloned sequence in quadrate box includes front circular frame, back circular frame of circRNA biogenesis (grey part) and full-length of 584-bp circPTK2 (red part). h , i Endogenous miR-429/miR-200b-3p levels in A549 and H226 cells transiently overexpressing circPTK2. * P
    Figure Legend Snippet: CircPTK2 binds directly to miR-429/miR-200b-3p in NSCLC cells. a Schematic description for the subcloning of the predicted miR-429/miR-200b-3p binding sites of circPTK2 exon11 in psiCHECK-2 luciferase vector. Predicted duplex formation between miR-429/miR-200b-3p and the wild-type/mutant of miR-429/miR-200b-3p binding sites of circPTK2 exon11 was shown. The entire subcloning sequences were listed in Additional file 4 : Table S2. b , c Relative luciferase activity of the wild-type/mutant circPTK2 exon11 reporter gene in A549 and H226 cells transfected with miR-429/miR-200b-3p or negative control (miR-NC). Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity was determined after normalizing against the firefly luciferase activity. d According to the flowchart outlining the experimental procedures ( left ), anti-AGO2 RIP was conducted in A549 cells transiently overexpressing miR-429/miR-200b-3p or miR-NC, and followed by RT-PCR and gel-staining analyses to detect circPTK2 enrichment ( right ). The 10% input was obtained as positive control before immunoprecipitation, and subjected to RT-PCR to confirm the presence of circPTK2. Anti-IgG antibody was used as a negative control. Red arrows represent divergent primers spanning the back-splice junction site of circPTK2. e According to the RNA pull-down flowchart ( left ), whole-cell lysates from A549 cells were incubated with biotinylated probes against circPTK2; after pull-down, endogenous circPTK2 ( middle ) and miR-429/miR-200b-3p ( right ) enrichments were detected by qRT-PCR. Results were presented as the percentage of pull-down to input. PD, pull-down. f Co-localization between circPTK2 (red) and miR-429/miR-200b-3p (green) was observed (arrowheads) by fluorescence in situ hybridization in A549 cells. Cell nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. g CircPTK2 expression in A549 and H226 cells transiently overexpressing circPTK2. Cells were transiently transfected with pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector ( upper panel ) for 48 h or 72 h and then subjected to qRT-PCR analysis ( bottom panel ). The empty vector was served as negative control. In the upper panel, the subcloned sequence in quadrate box includes front circular frame, back circular frame of circRNA biogenesis (grey part) and full-length of 584-bp circPTK2 (red part). h , i Endogenous miR-429/miR-200b-3p levels in A549 and H226 cells transiently overexpressing circPTK2. * P

    Techniques Used: Subcloning, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Negative Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Staining, Positive Control, Immunoprecipitation, Incubation, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Expressing

    miR-429 enhances TGF-β-induced EMT and invasion in NSCLC cells. a After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Western blot analysis was performed to examine the expression of N-cadherin, which was normalized to β-actin. c A549 and H226 cells transiently overexpressing miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. d Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. e After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. qRT-PCR analysis was done to determine the relative Snail mRNA expression. f After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. N-cadherin expression was analyzed by western blot. g A549 and H226 cells transiently overexpressing anti-miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. h Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. ** P
    Figure Legend Snippet: miR-429 enhances TGF-β-induced EMT and invasion in NSCLC cells. a After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. Snail mRNA expression was quantified by qRT-PCR analysis. Snail mRNA level of the unstimulated cells was assigned the value 1, and the relative Snail mRNA expression in TGF-β1-stimulated cells was recalculated accordingly. b After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. Western blot analysis was performed to examine the expression of N-cadherin, which was normalized to β-actin. c A549 and H226 cells transiently overexpressing miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. d Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. e After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 1 h and 2 h, respectively. qRT-PCR analysis was done to determine the relative Snail mRNA expression. f After being serum-starved for 24 h, A549 and H226 cells transiently overexpressing anti-miR-429 were treated with or without TGF-β1 (5 ng/ml) for 24 h and 48 h, respectively. N-cadherin expression was analyzed by western blot. g A549 and H226 cells transiently overexpressing anti-miR-429 were treated as above and allowed to migrate through an 8-μM pore in transwells. Migrated cells were stained and counted in at least three light microscopic fields. Scale bar, 100 μm. h Cells were treated as above and allowed to invade through Matrigel-coated membrane in transwells. Invasive cells were stained and counted under a light microscope. Scale bar, 100 μm. ** P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Staining, Light Microscopy

    miR-429 represses TIF1γ expression by targeting 3’-UTR of TIF1γ transcript. a Schematic description for the subcloning of the predicted miR-429 binding sites of TIF1γ 3’-UTR in psiCHECK-2 luciferase vector. Predicted duplex formation between miR-429 and the wild-type/mutant of miR-429 binding sites was indicated. The entire subcloning sequences were listed in Additional file 4 : Table S2. b Relative luciferase activity of the wild-type/mutant TIF1γ 3’-UTR reporter gene in A549 and H226 cells transfected with miR-429 or negative control (miR-NC). Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity was determined after normalizing against the firefly luciferase activity. c qRT-PCR analysis of miR-429 expression levels in A549 and H226 cells transfected with miR-429 mimics or miR-NC. U6 was employed as internal control. d , e TIF1γ mRNA and protein expression in A549 and H226 cells transfected with miR-429 mimics or miR-NC. β-actin was used as internal control. f miR-429 expression levels in A549 and H226 cells transfected with miR-429 inhibitors (anti-miR-429) or negative control (anti-miR-NC). Scrambled sequence was used as anti-miR-NC. g , h TIF1γ mRNA and protein expression in A549 and H226 cells transfected with anti-miR-429 or anti-miR-NC. * P
    Figure Legend Snippet: miR-429 represses TIF1γ expression by targeting 3’-UTR of TIF1γ transcript. a Schematic description for the subcloning of the predicted miR-429 binding sites of TIF1γ 3’-UTR in psiCHECK-2 luciferase vector. Predicted duplex formation between miR-429 and the wild-type/mutant of miR-429 binding sites was indicated. The entire subcloning sequences were listed in Additional file 4 : Table S2. b Relative luciferase activity of the wild-type/mutant TIF1γ 3’-UTR reporter gene in A549 and H226 cells transfected with miR-429 or negative control (miR-NC). Scrambled sequence was used as miR-NC. Relative Renilla luciferase activity was determined after normalizing against the firefly luciferase activity. c qRT-PCR analysis of miR-429 expression levels in A549 and H226 cells transfected with miR-429 mimics or miR-NC. U6 was employed as internal control. d , e TIF1γ mRNA and protein expression in A549 and H226 cells transfected with miR-429 mimics or miR-NC. β-actin was used as internal control. f miR-429 expression levels in A549 and H226 cells transfected with miR-429 inhibitors (anti-miR-429) or negative control (anti-miR-NC). Scrambled sequence was used as anti-miR-NC. g , h TIF1γ mRNA and protein expression in A549 and H226 cells transfected with anti-miR-429 or anti-miR-NC. * P

    Techniques Used: Expressing, Subcloning, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Negative Control, Sequencing, Quantitative RT-PCR

    2) Product Images from "Health hazards of methylammonium lead iodide based perovskites: cytotoxicity studies hazards of methylammonium lead iodide based perovskites: cytotoxicity studies †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tx00303b"

    Article Title: Health hazards of methylammonium lead iodide based perovskites: cytotoxicity studies hazards of methylammonium lead iodide based perovskites: cytotoxicity studies †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tx00303b

    Journal: Toxicology Research

    doi: 10.1039/c5tx00303b

    Morphological and proliferation changes caused by MAPbI 3 in A549 human lung epithelial cells. Cells were treated over 5 days with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and the cellular changes were evaluated using complementary assays. (a) Variation of the cellular density was evaluated using a Neubauer hemocytometer; (b) morphological changes observed by bright field optical microscopy were quantified then normalized to non-treated cells, which have an overall triangle-shaped form and a size between 15 and 20 μm. The size significantly increased (“giant cells”, size > 50 μm) in treated cells and became round. Some cells acquired a stretched/elongated morphology (“elongated cells”, width
    Figure Legend Snippet: Morphological and proliferation changes caused by MAPbI 3 in A549 human lung epithelial cells. Cells were treated over 5 days with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and the cellular changes were evaluated using complementary assays. (a) Variation of the cellular density was evaluated using a Neubauer hemocytometer; (b) morphological changes observed by bright field optical microscopy were quantified then normalized to non-treated cells, which have an overall triangle-shaped form and a size between 15 and 20 μm. The size significantly increased (“giant cells”, size > 50 μm) in treated cells and became round. Some cells acquired a stretched/elongated morphology (“elongated cells”, width

    Techniques Used: Microscopy

    Ultrastructural analysis of A549 cells. (a) Low magnification electron micrograph of the non-treated A549 cells grown in a monolayer on the plastic Thermanox coverslip and processed for the electron microscopy. A typical cellular morphology is shown with single nucleus (blue) well preserved nuclear membrane and multiple nucleoli and lamellar bodies (LB; white arrows); (b) changed morphology of the A549 cells upon the treatment with MAPbI 3 . Low magnification overview image of the large “giant” multinucleated cell containing 7 nuclei and significantly increased number of lamellar bodies depicted by white arrows; (c) two characteristic higher magnification images of the normal mitochondrial (green) morphology with typical cristae organization, tubular organization of the endoplasmic reticulum (black arrowheads) together with lamellar bodies (white arrows); (d) two characteristic higher magnification images demonstrating the severe damage of the mitochondria, particularly increased their size compared to the non-treated A549 cells and highly dilated intra-mitochondrial space and reduced cristae organization. The lamellar bodies (white arrows) and tubular organization of the endoplasmic reticulum are marked (black arrowheads). Scale bars: 5 μm in (a) and (b); 1 μm in (c) and (d).
    Figure Legend Snippet: Ultrastructural analysis of A549 cells. (a) Low magnification electron micrograph of the non-treated A549 cells grown in a monolayer on the plastic Thermanox coverslip and processed for the electron microscopy. A typical cellular morphology is shown with single nucleus (blue) well preserved nuclear membrane and multiple nucleoli and lamellar bodies (LB; white arrows); (b) changed morphology of the A549 cells upon the treatment with MAPbI 3 . Low magnification overview image of the large “giant” multinucleated cell containing 7 nuclei and significantly increased number of lamellar bodies depicted by white arrows; (c) two characteristic higher magnification images of the normal mitochondrial (green) morphology with typical cristae organization, tubular organization of the endoplasmic reticulum (black arrowheads) together with lamellar bodies (white arrows); (d) two characteristic higher magnification images demonstrating the severe damage of the mitochondria, particularly increased their size compared to the non-treated A549 cells and highly dilated intra-mitochondrial space and reduced cristae organization. The lamellar bodies (white arrows) and tubular organization of the endoplasmic reticulum are marked (black arrowheads). Scale bars: 5 μm in (a) and (b); 1 μm in (c) and (d).

    Techniques Used: Electron Microscopy

    Effect of MAPbI 3 on the mitochondrial function of A549 cells. A549 cells were treated with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and their mitochondrial activity was evaluated. (a) MTT assay after 1, 3, 5 and 7 days of exposure to MAPbI 3 showing increasing metabolic activity normalized to the number of counted cells; (b) mitochondrial membrane potential (Δ Ψ m ) was assessed in non-treated and treated cells (100 μg ml –1 of MAPbI 3 ) at 5 days post-treatment. Cells were stained by Mitotracker Red CMXRos and analyzed by flow cytometry. These results corroborate the increased mitochondrial activity seen in (a); (c) the overall mass of mitochondria was measured in non-treated and treated cells (100 μg ml –1 of MAPbI 3 ) at 5 days post-treatment. Cells were stained by Mitotracker Green and analyzed by flow cytometry. All the histograms show an average of at least 3 independent repeats (each condition in triplicate). Bars are means ± S.D. One-way ANOVA test followed by a Tukey–Kramer post-hoc test were performed (non-treated vs. MAPbI 3 treated conditions), * p
    Figure Legend Snippet: Effect of MAPbI 3 on the mitochondrial function of A549 cells. A549 cells were treated with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and their mitochondrial activity was evaluated. (a) MTT assay after 1, 3, 5 and 7 days of exposure to MAPbI 3 showing increasing metabolic activity normalized to the number of counted cells; (b) mitochondrial membrane potential (Δ Ψ m ) was assessed in non-treated and treated cells (100 μg ml –1 of MAPbI 3 ) at 5 days post-treatment. Cells were stained by Mitotracker Red CMXRos and analyzed by flow cytometry. These results corroborate the increased mitochondrial activity seen in (a); (c) the overall mass of mitochondria was measured in non-treated and treated cells (100 μg ml –1 of MAPbI 3 ) at 5 days post-treatment. Cells were stained by Mitotracker Green and analyzed by flow cytometry. All the histograms show an average of at least 3 independent repeats (each condition in triplicate). Bars are means ± S.D. One-way ANOVA test followed by a Tukey–Kramer post-hoc test were performed (non-treated vs. MAPbI 3 treated conditions), * p

    Techniques Used: Activity Assay, MTT Assay, Staining, Flow Cytometry, Cytometry

    MAPbI 3 does not induce cell demise in A549 human lung epithelial cells. The cells were treated with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and the cellular toxicity was evaluated using flow cytometry (a). A549 cells were harvested after 1, 3, 5 or 7 days post-treatment and stained with SG. The cell death level is expressed as the percentage of cells with a compromised cell membrane (SG positive cells) to the total cell number. (b) A549 cells were harvested 5 days post-treatment and caspase 3, caspase 9 and caspase 8 activities were measured using fluorogenic assays. All the histograms show an average of at least 3 independent repeats (each condition in triplicate). No significance was observed.
    Figure Legend Snippet: MAPbI 3 does not induce cell demise in A549 human lung epithelial cells. The cells were treated with increasing concentrations of MAPbI 3 (50, 100 and 200 μg ml –1 ) and the cellular toxicity was evaluated using flow cytometry (a). A549 cells were harvested after 1, 3, 5 or 7 days post-treatment and stained with SG. The cell death level is expressed as the percentage of cells with a compromised cell membrane (SG positive cells) to the total cell number. (b) A549 cells were harvested 5 days post-treatment and caspase 3, caspase 9 and caspase 8 activities were measured using fluorogenic assays. All the histograms show an average of at least 3 independent repeats (each condition in triplicate). No significance was observed.

    Techniques Used: Flow Cytometry, Cytometry, Staining

    3) Product Images from "Requirement of a Novel Splicing Variant of Human Histone Deacetylase 6 for TGF-?1-mediated Gene Activation"

    Article Title: Requirement of a Novel Splicing Variant of Human Histone Deacetylase 6 for TGF-?1-mediated Gene Activation

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2010.01.091

    Manifestation of the p114 protein. A) Comparison of the structure of the conceptual proteins of p131 and p114. B) Expression of potential isoforms of the hHDAC6 proteins and acetylated α-tubulin was examined in A549 cells stably expressing either scrambled (sc) or hHDAC6-targeting siRNA (HD6) using immunoblots. C) A549 and MCF-7 cells were transfected with either scrambled (−) or p114-targeting siRNA (+). The protein levels of p131, p114, and acetylated α-tubulin were examined using immunoblots. D) A549 cells were transfected with either the backbone or p114/p131 expression vectors. The protein levels of p131, p114, and acetylated α-tubulin were evaluated using immunoblots. The results are representative of at least 2 independent experiments.
    Figure Legend Snippet: Manifestation of the p114 protein. A) Comparison of the structure of the conceptual proteins of p131 and p114. B) Expression of potential isoforms of the hHDAC6 proteins and acetylated α-tubulin was examined in A549 cells stably expressing either scrambled (sc) or hHDAC6-targeting siRNA (HD6) using immunoblots. C) A549 and MCF-7 cells were transfected with either scrambled (−) or p114-targeting siRNA (+). The protein levels of p131, p114, and acetylated α-tubulin were examined using immunoblots. D) A549 cells were transfected with either the backbone or p114/p131 expression vectors. The protein levels of p131, p114, and acetylated α-tubulin were evaluated using immunoblots. The results are representative of at least 2 independent experiments.

    Techniques Used: Expressing, Stable Transfection, Western Blot, Transfection

    Manifestation of the p114 mRNA. A) The schemes to amplify p131 and p114. The shaded boxes indicate exons and the connecting lines indicate introns. B) Total cell RNA was extracted from A549 (A) and MCF-7 (M) cells. The p131 and p114 mRNAs were detected by RT-PCR using a pair of non-selective primers. C) Similar to B except that cDNA was amplified using a pair of the p114 mRNA-specific primers. D) The mRNA levels of p114 (p114), and p114 and p131 combined (p114+p131) were determined using quantitative RT-PCR. Ratios of p114/(p114+p131) were obtained after normalization to the Ct values of the housekeeping gene 36B4. Mean and standard deviations were obtained from 3 independent experiments. *, P value
    Figure Legend Snippet: Manifestation of the p114 mRNA. A) The schemes to amplify p131 and p114. The shaded boxes indicate exons and the connecting lines indicate introns. B) Total cell RNA was extracted from A549 (A) and MCF-7 (M) cells. The p131 and p114 mRNAs were detected by RT-PCR using a pair of non-selective primers. C) Similar to B except that cDNA was amplified using a pair of the p114 mRNA-specific primers. D) The mRNA levels of p114 (p114), and p114 and p131 combined (p114+p131) were determined using quantitative RT-PCR. Ratios of p114/(p114+p131) were obtained after normalization to the Ct values of the housekeeping gene 36B4. Mean and standard deviations were obtained from 3 independent experiments. *, P value

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

    Impact of p114-targeting RNAi on TGF-β1-induced deacetylation of α-tubulin. A) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of p114 (p114) and the combined p114 and p131 were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. B) The culture conditions were similar to part A except that the protein levels of p131, p114, and acetylated α-tubulin were determined using immunoblots. Mean and standard deviations were obtained from 4 independent transfections.
    Figure Legend Snippet: Impact of p114-targeting RNAi on TGF-β1-induced deacetylation of α-tubulin. A) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of p114 (p114) and the combined p114 and p131 were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. B) The culture conditions were similar to part A except that the protein levels of p131, p114, and acetylated α-tubulin were determined using immunoblots. Mean and standard deviations were obtained from 4 independent transfections.

    Techniques Used: Isolation, Transfection, Quantitative RT-PCR, Western Blot

    Requirement of p114 for TGF-β1-activated gene expression. A-D) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or hHDAC6p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of PAI-1 (A), PDGF-B (B), Collagen I (C), and E-cadherin (D) were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. E) A549 cells were co-transfected with luciferase reporter constructs and siRNAs followed by exposure to TGF-β1 for 24 hrs. The ratios of TGF-β1 responsive 3TP-LUX activity over the co-transfected RL-TK activity were compared across the groups. Mean and standard deviations were obtained from 4 independent transfections. *, P value
    Figure Legend Snippet: Requirement of p114 for TGF-β1-activated gene expression. A-D) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or hHDAC6p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of PAI-1 (A), PDGF-B (B), Collagen I (C), and E-cadherin (D) were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. E) A549 cells were co-transfected with luciferase reporter constructs and siRNAs followed by exposure to TGF-β1 for 24 hrs. The ratios of TGF-β1 responsive 3TP-LUX activity over the co-transfected RL-TK activity were compared across the groups. Mean and standard deviations were obtained from 4 independent transfections. *, P value

    Techniques Used: Expressing, Isolation, Transfection, Quantitative RT-PCR, Luciferase, Construct, Activity Assay

    4) Product Images from "Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6"

    Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6

    Journal: Virology Journal

    doi: 10.1186/1743-422X-10-207

    Overexpression of Myc-PRMT6 does not affect basal transcription from the HIV-1 LTR promoter. A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transcriptional reporter plasmid with varying amounts of Myc-PRMT6 plasmid, as indicated. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample. Columns represent the means and standard deviations of three independent experiments.
    Figure Legend Snippet: Overexpression of Myc-PRMT6 does not affect basal transcription from the HIV-1 LTR promoter. A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transcriptional reporter plasmid with varying amounts of Myc-PRMT6 plasmid, as indicated. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample. Columns represent the means and standard deviations of three independent experiments.

    Techniques Used: Over Expression, Transfection, Luciferase, Plasmid Preparation

    HIV-1 produced in A549 cells overexpressing Myc-PRMT6 are competent for infectivity. ( A ) A549 cells were transfected with a HIV-1 proviral plasmid in which the EGFP gene had been inserted into the nef reading frame, along with a plasmid expressing the VSV-G envelope glycoprotein to enable pseudotyping. Cells were simultaneously cotransfected with either empty vector (-), the wild type Myc-PRMT6 plasmid (WT) or a methyltransferase-inactive mutant Myc-PRMT6 plasmid (Mut.). Western blotting was performed on producer cell lysates using an anti-PRMT6 antibody. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( B ) HeLa and A549 target cells were infected with the pseudotyped viruses collected from the A549 producer cells in A after normalizing for capsid levels. EGFP expression in the target cells, which indicates successful infection, was then quantified by flow cytometry. A representative dot plot of target cells infected with virus from the empty vector-cotransfected producer cells is shown, in which GFP-positive, phycoerythrin (PE)-negative cells are gated (green). ( C ) The proportions of GFP-positive HeLa (black columns) and A549 (white columns) target cells were quantified for each infection sample and are shown arranged by the Myc-PRMT6 plasmid cotransfected in the virus producer cells. Data are expressed relative to the respective empty vector sample (“None”), and columns represent the means and standard deviations of two independent infections using independent virus stocks.
    Figure Legend Snippet: HIV-1 produced in A549 cells overexpressing Myc-PRMT6 are competent for infectivity. ( A ) A549 cells were transfected with a HIV-1 proviral plasmid in which the EGFP gene had been inserted into the nef reading frame, along with a plasmid expressing the VSV-G envelope glycoprotein to enable pseudotyping. Cells were simultaneously cotransfected with either empty vector (-), the wild type Myc-PRMT6 plasmid (WT) or a methyltransferase-inactive mutant Myc-PRMT6 plasmid (Mut.). Western blotting was performed on producer cell lysates using an anti-PRMT6 antibody. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( B ) HeLa and A549 target cells were infected with the pseudotyped viruses collected from the A549 producer cells in A after normalizing for capsid levels. EGFP expression in the target cells, which indicates successful infection, was then quantified by flow cytometry. A representative dot plot of target cells infected with virus from the empty vector-cotransfected producer cells is shown, in which GFP-positive, phycoerythrin (PE)-negative cells are gated (green). ( C ) The proportions of GFP-positive HeLa (black columns) and A549 (white columns) target cells were quantified for each infection sample and are shown arranged by the Myc-PRMT6 plasmid cotransfected in the virus producer cells. Data are expressed relative to the respective empty vector sample (“None”), and columns represent the means and standard deviations of two independent infections using independent virus stocks.

    Techniques Used: Produced, Infection, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Luciferase, Flow Cytometry, Cytometry

    The A549 cell line does not express detectable levels of PRMT6 protein. ( A ) Western blot of cell lysates from the A549, BJAB and HeLa cell lines detected with anti-PRMT6, anti-PRMT1 and anti-β-tubulin antibodies as indicated. ( B ) Relative expression ratios of PRMT1 and PRMT6 mRNA transcripts in A549 versus HeLa cells. Total RNA were extracted from A549 and HeLa cells before being reverse transcribed into cDNA using random primers. Quantitative PCR was then performed on the cDNA samples using primers specific for PRMT1 , PRMT6 and GAPDH transcripts. Relative expression ratios for PRMT1 and PRMT6 were calculated according to the method of [ 29 ]. ( C ) Ectopic Myc-PRMT6 increases the steady state levels of Tat-FLAG protein in A549 cells. Western blotting was performed on A549 cells transfected to express Tat-FLAG with (+) or without (-) Myc-PRMT6. Proteins were detected with anti-FLAG and anti-PRMT6 antibodies, respectively. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( D ) A549 cells transfected to express Tat-FLAG with or without Myc-PRMT6 were treated with cycloheximide and harvested at 0, 1, 2, 4, 6 and 8 h post-treatment. Western blotting was performed on total protein-equalized lysates. β-tubulin levels demonstrate equal sample loadings. ( E ) The Tat-FLAG band intensities in panel D were quantified, and their natural log values were plotted as a function of time. Values for Tat-FLAG co expressed with Myc-PRMT6 are indicated by the black boxes and values for Tat-FLAG expressed alone by the white boxes. The calculated Tat-FLAG protein half-lives are shown in the inset.
    Figure Legend Snippet: The A549 cell line does not express detectable levels of PRMT6 protein. ( A ) Western blot of cell lysates from the A549, BJAB and HeLa cell lines detected with anti-PRMT6, anti-PRMT1 and anti-β-tubulin antibodies as indicated. ( B ) Relative expression ratios of PRMT1 and PRMT6 mRNA transcripts in A549 versus HeLa cells. Total RNA were extracted from A549 and HeLa cells before being reverse transcribed into cDNA using random primers. Quantitative PCR was then performed on the cDNA samples using primers specific for PRMT1 , PRMT6 and GAPDH transcripts. Relative expression ratios for PRMT1 and PRMT6 were calculated according to the method of [ 29 ]. ( C ) Ectopic Myc-PRMT6 increases the steady state levels of Tat-FLAG protein in A549 cells. Western blotting was performed on A549 cells transfected to express Tat-FLAG with (+) or without (-) Myc-PRMT6. Proteins were detected with anti-FLAG and anti-PRMT6 antibodies, respectively. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. ( D ) A549 cells transfected to express Tat-FLAG with or without Myc-PRMT6 were treated with cycloheximide and harvested at 0, 1, 2, 4, 6 and 8 h post-treatment. Western blotting was performed on total protein-equalized lysates. β-tubulin levels demonstrate equal sample loadings. ( E ) The Tat-FLAG band intensities in panel D were quantified, and their natural log values were plotted as a function of time. Values for Tat-FLAG co expressed with Myc-PRMT6 are indicated by the black boxes and values for Tat-FLAG expressed alone by the white boxes. The calculated Tat-FLAG protein half-lives are shown in the inset.

    Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase

    Overexpression of Myc-PRMT6 does not alter Tat-FLAG mediated transactivation in either A549 or HeLa cells. ( A ) A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transactivation reporter plasmid along with varying amounts of Tat-FLAG plasmid, as indicated, and either with (black columns) or without (white columns) Myc-PRMT6 plasmid. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency and assay variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample expressed relative to the “0 ng Tat-FLAG” sample. Columns represent the means and standard deviations of four independent experiments. ( B ) The same experiment as in A was performed in HeLa cells. Columns represent the means and standard deviations of three independent experiments. ( C ) Overexpression of PRMT6 does not alter overall plasmid expression. The Renilla luciferase activities from A were normalized to total lysate protein concentrations before being expressed relative to the “0 ng Tat-FLAG without Myc-PRMT6” sample. Samples in which Myc-PRMT6 plasmid was (+) or was not (-) cotransfected are indicated. Columns represent the means and standard deviations of four independent experiments. ( D ) The same determinations as in C were performed with the samples from B . Columns represent the means and standard deviations of three independent experiments.
    Figure Legend Snippet: Overexpression of Myc-PRMT6 does not alter Tat-FLAG mediated transactivation in either A549 or HeLa cells. ( A ) A549 cells were transfected with a long terminal repeat (LTR)- Photinus luciferase transactivation reporter plasmid along with varying amounts of Tat-FLAG plasmid, as indicated, and either with (black columns) or without (white columns) Myc-PRMT6 plasmid. All samples were cotransfected with a SV40-promotered Renilla luciferase plasmid to control for transfection efficiency and assay variations. Transfected cells were lysed and assayed for both Photinus and Renilla luciferase activities, the ratio of which is shown for each sample expressed relative to the “0 ng Tat-FLAG” sample. Columns represent the means and standard deviations of four independent experiments. ( B ) The same experiment as in A was performed in HeLa cells. Columns represent the means and standard deviations of three independent experiments. ( C ) Overexpression of PRMT6 does not alter overall plasmid expression. The Renilla luciferase activities from A were normalized to total lysate protein concentrations before being expressed relative to the “0 ng Tat-FLAG without Myc-PRMT6” sample. Samples in which Myc-PRMT6 plasmid was (+) or was not (-) cotransfected are indicated. Columns represent the means and standard deviations of four independent experiments. ( D ) The same determinations as in C were performed with the samples from B . Columns represent the means and standard deviations of three independent experiments.

    Techniques Used: Over Expression, Transfection, Luciferase, Plasmid Preparation, Expressing

    5) Product Images from "Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells"

    Article Title: Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14047273

    Digoxin suppresses mRNA and protein expressions of HIF-1α and reduces the HIF-1/DNA complex in A549 cells under hypoxic conditions. ( a ) Under normoxic or hypoxic conditions, mRNA and protein levels of HIF-1α in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were determined. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin suppresses mRNA and protein expressions of HIF-1α and reduces the HIF-1/DNA complex in A549 cells under hypoxic conditions. ( a ) Under normoxic or hypoxic conditions, mRNA and protein levels of HIF-1α in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were determined. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used:

    Digoxin inhibits mRNA and protein expression of NDRG1 in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of NDRG1 in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were measured. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin inhibits mRNA and protein expression of NDRG1 in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of NDRG1 in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were measured. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used: Expressing

    Digoxin inhibits the viability of A549 cells under hypoxic conditions. A549 cell viability was measured by MTT assay and analyzed at different time points (0, 24, 48 and 72 h) with or without various concentrations of digoxin (0.01, 0.1, and 1 μM). Quantitative data are presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin inhibits the viability of A549 cells under hypoxic conditions. A549 cell viability was measured by MTT assay and analyzed at different time points (0, 24, 48 and 72 h) with or without various concentrations of digoxin (0.01, 0.1, and 1 μM). Quantitative data are presented as mean ± SD ( n = 4). * p

    Techniques Used: MTT Assay

    Digoxin attenuates mRNA and protein expression of VEGF in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of VEGF in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were analyzed. Quantitative data were presented as mean ± SD ( n = 4). * p
    Figure Legend Snippet: Digoxin attenuates mRNA and protein expression of VEGF in A549 cells under hypoxic conditions. Under normoxic or hypoxic conditions, mRNA and protein levels of VEGF in A549 cells treated with or without various concentrations of digoxin (0.01, 0.1, and 1 μM) for 24 h were analyzed. Quantitative data were presented as mean ± SD ( n = 4). * p

    Techniques Used: Expressing

    6) Product Images from "PAK1 Kinase Promotes Cell Motility and Invasiveness through CRK-II Serine Phosphorylation in Non-Small Cell Lung Cancer Cells"

    Article Title: PAK1 Kinase Promotes Cell Motility and Invasiveness through CRK-II Serine Phosphorylation in Non-Small Cell Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042012

    A-Wound healing assays in A549 cells stably expressing wild type CRK-II (WT) or CRK-II phosphomimetic (Ser41Asp) mutants following siRNA mediated PAK1 silencing. Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after the establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P
    Figure Legend Snippet: A-Wound healing assays in A549 cells stably expressing wild type CRK-II (WT) or CRK-II phosphomimetic (Ser41Asp) mutants following siRNA mediated PAK1 silencing. Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after the establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P

    Techniques Used: Stable Transfection, Expressing

    A-Relative p120-catenin (CTNND1) promoter activity in A549 cells following siRNA mediated silencing of PAK1 and PAK2. B- Quantitative real time PCR measurement of PAK1 and PAK2 mRNA in order to determine the silencing efficiency of siRNA. C-Western blots showing phospho-serine 41 CRK-II and p120-catenin expression following siRNA mediated PAK1 silencing in A549 and H157 cells. D- Pak1 phosphorylation consensus sequence in several Pak1 substrates. Residues that are phosphorylated by Pak1 are highlighted in gray. Circled are the upstream arginine residues that are important for Pak1 target phosphorylation.
    Figure Legend Snippet: A-Relative p120-catenin (CTNND1) promoter activity in A549 cells following siRNA mediated silencing of PAK1 and PAK2. B- Quantitative real time PCR measurement of PAK1 and PAK2 mRNA in order to determine the silencing efficiency of siRNA. C-Western blots showing phospho-serine 41 CRK-II and p120-catenin expression following siRNA mediated PAK1 silencing in A549 and H157 cells. D- Pak1 phosphorylation consensus sequence in several Pak1 substrates. Residues that are phosphorylated by Pak1 are highlighted in gray. Circled are the upstream arginine residues that are important for Pak1 target phosphorylation.

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Sequencing

    A-Wound healing assays in A549 cells stably expressing pCMV vector, wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P
    Figure Legend Snippet: A-Wound healing assays in A549 cells stably expressing pCMV vector, wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. Endogenous CRK is silenced in all conditions by siRNA. B- Measurement of wound surface area 24 hours after establishment of the wound among the above mentioned groups. The average is calculated following measurement of three separate experiments. (2 tailed student’s t-test: ** P

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation

    A- Relative p120-catenin (CTNND1) promoter activity in A549, Rh2 and H157 cell lines following transient transfection of CRK-II, CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. (2 tailed student’s t-test: * P
    Figure Legend Snippet: A- Relative p120-catenin (CTNND1) promoter activity in A549, Rh2 and H157 cell lines following transient transfection of CRK-II, CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants. (2 tailed student’s t-test: * P

    Techniques Used: Activity Assay, Transfection

    Relative p120-catenin (CTNND1) promoter activity in A549 cells stably expressing wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants following siRNA mediated silencing of PAK1. Endogenous CRK is silenced in all conditions by siRNA. (2 tailed student’s t-test: * P
    Figure Legend Snippet: Relative p120-catenin (CTNND1) promoter activity in A549 cells stably expressing wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants following siRNA mediated silencing of PAK1. Endogenous CRK is silenced in all conditions by siRNA. (2 tailed student’s t-test: * P

    Techniques Used: Activity Assay, Stable Transfection, Expressing

    7) Product Images from "Inhibition of CK2? Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-Like Side Population in Human Lung Cancer Cells"

    Article Title: Inhibition of CK2? Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-Like Side Population in Human Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038996

    CK2α Knockdown Down-Regulates Hh Signal Pathway Transduction and Reduces SP Proportion in A549. (A) Hh downstream genes expression detected by real-time RT-PCR. The mRNA level of the four Hh target genes (Gli1, Ptc1, Ptc2 and Cyclin E1) decreased significantly after CK2α knockdown. * P
    Figure Legend Snippet: CK2α Knockdown Down-Regulates Hh Signal Pathway Transduction and Reduces SP Proportion in A549. (A) Hh downstream genes expression detected by real-time RT-PCR. The mRNA level of the four Hh target genes (Gli1, Ptc1, Ptc2 and Cyclin E1) decreased significantly after CK2α knockdown. * P

    Techniques Used: Transduction, Expressing, Quantitative RT-PCR

    CK2α Inhibition Down-Regulates Gli1 Expression and Transcriptional Activity. (A) Quantitative Gli1 mRNA levels after treatment with CK2 subunit-specific siRNA detected by real-time RT-PCR. Silencing of CK2α significantly reduced Gli1 mRNA levels both in A549 and H1299 cell lines (by 50% and 45%, respectively). Silencing of CK2β also resulted in a significant decrease of Gli1 in both cell lines. Minimal Gli1 mRNA level was noticed in the normal lung (NL). * P
    Figure Legend Snippet: CK2α Inhibition Down-Regulates Gli1 Expression and Transcriptional Activity. (A) Quantitative Gli1 mRNA levels after treatment with CK2 subunit-specific siRNA detected by real-time RT-PCR. Silencing of CK2α significantly reduced Gli1 mRNA levels both in A549 and H1299 cell lines (by 50% and 45%, respectively). Silencing of CK2β also resulted in a significant decrease of Gli1 in both cell lines. Minimal Gli1 mRNA level was noticed in the normal lung (NL). * P

    Techniques Used: Inhibition, Expressing, Activity Assay, Quantitative RT-PCR

    8) Product Images from "Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549"

    Article Title: Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-28-16

    Analysis of differentially expressed proteins by 2-DE (two-dimensional electrophoresis) . Two-dimensional electrophoresis based proteomics approaches were performed to determine the proteins expressed differently. Representative 2-DE gels of Eahy926 and A549 cells. Differential expression protein spots were labeled with numbers.
    Figure Legend Snippet: Analysis of differentially expressed proteins by 2-DE (two-dimensional electrophoresis) . Two-dimensional electrophoresis based proteomics approaches were performed to determine the proteins expressed differently. Representative 2-DE gels of Eahy926 and A549 cells. Differential expression protein spots were labeled with numbers.

    Techniques Used: Electrophoresis, Expressing, Labeling

    Western blot analysis of Hsp60 . Western blot was performed to verify the expression of HSP60 in A549 and Eahy926 cells. The expression of HSP60 in A549 cells was higher than that in Eahy926 cells.
    Figure Legend Snippet: Western blot analysis of Hsp60 . Western blot was performed to verify the expression of HSP60 in A549 and Eahy926 cells. The expression of HSP60 in A549 cells was higher than that in Eahy926 cells.

    Techniques Used: Western Blot, Expressing

    Adhesion of Eahy926 and A549 cells with Matrigel in vitro . (A) For adhesion test, extracellular matrix (Matrigel) was used. Representative images of Eahy926 and A549 cells adhered with the Matrigel after incubation for 1 h; (B) Number of adhesive cells with extracellular matrix (Matrigel) was measured by MTT assays. The difference in adhesion ability between Eahy926 and A549 cells was shown as OD value (OD: optical density). Independent experiments were measured in triplicate and repeated three times for each cell type; Columns, mean of independent experiments measured in triplicate and repeated for three independent times; bars, SD (P
    Figure Legend Snippet: Adhesion of Eahy926 and A549 cells with Matrigel in vitro . (A) For adhesion test, extracellular matrix (Matrigel) was used. Representative images of Eahy926 and A549 cells adhered with the Matrigel after incubation for 1 h; (B) Number of adhesive cells with extracellular matrix (Matrigel) was measured by MTT assays. The difference in adhesion ability between Eahy926 and A549 cells was shown as OD value (OD: optical density). Independent experiments were measured in triplicate and repeated three times for each cell type; Columns, mean of independent experiments measured in triplicate and repeated for three independent times; bars, SD (P

    Techniques Used: In Vitro, Incubation, MTT Assay

    Migration and invasion of Eahy926 and A549 cells with transwell chambers in vitro . (A) Cell migration was evaluated by Milliwell assays. Cells migrating to the lower surface of filters were stained with hematoxylin solution. Representative images of Eahy926 and A549 cells on the lower side of a membrane after incubation for 6 h; (B) The difference in migration ability between Eahy926 and A549 cells; Columns, mean of independent experiments measured in triplicate and repeated for three independent times; bars, SD (P
    Figure Legend Snippet: Migration and invasion of Eahy926 and A549 cells with transwell chambers in vitro . (A) Cell migration was evaluated by Milliwell assays. Cells migrating to the lower surface of filters were stained with hematoxylin solution. Representative images of Eahy926 and A549 cells on the lower side of a membrane after incubation for 6 h; (B) The difference in migration ability between Eahy926 and A549 cells; Columns, mean of independent experiments measured in triplicate and repeated for three independent times; bars, SD (P

    Techniques Used: Migration, In Vitro, Staining, Incubation

    Tumorigenicity of Eahy926 and A549 cells in vivo . (A) No tumor mass formed roughly within 14 days after s.c. injection of Eahy926 cells; (B) Tumor mass formed roughly within 10 days after s.c. injection of A549 cells; (C) On day 14 after s.c inoculation of Eahy926 cells; tissues collected from the inoculative site were identified as inflammatory necrosis in the Eahy926 cells group; (D) On day 14 after s.c inoculation of A549 cells, classic tumor microstructure was found in the A549 cells group and the rate of tumorigenicity was 100%.
    Figure Legend Snippet: Tumorigenicity of Eahy926 and A549 cells in vivo . (A) No tumor mass formed roughly within 14 days after s.c. injection of Eahy926 cells; (B) Tumor mass formed roughly within 10 days after s.c. injection of A549 cells; (C) On day 14 after s.c inoculation of Eahy926 cells; tissues collected from the inoculative site were identified as inflammatory necrosis in the Eahy926 cells group; (D) On day 14 after s.c inoculation of A549 cells, classic tumor microstructure was found in the A549 cells group and the rate of tumorigenicity was 100%.

    Techniques Used: In Vivo, Injection

    Close-up image of partial differential expression of protein spots between Eahy926 and A549 cells . Protein spot discrepancies were arrowed and marked with number. Each bar graph showed expression level of protein spots in Eahy926 and A549 cells.
    Figure Legend Snippet: Close-up image of partial differential expression of protein spots between Eahy926 and A549 cells . Protein spot discrepancies were arrowed and marked with number. Each bar graph showed expression level of protein spots in Eahy926 and A549 cells.

    Techniques Used: Expressing

    Proliferation and cell cycle of Eahy926 and A549 cells . (A) Cells number was counted after trypsinization every 24 hours to draw the growth curves of Eahy926 cells and A549 cells (P > 0.1); (B and C) Cell cycle analysis was performed on FACSCalibur flow cytometer. The percentages of cell population in subG 1 , G 1 , S or G 2 /M phases were calculated from histograms by using the CellQuest software; The data represent the mean ± SD of three independent experiments (P > 0.05).
    Figure Legend Snippet: Proliferation and cell cycle of Eahy926 and A549 cells . (A) Cells number was counted after trypsinization every 24 hours to draw the growth curves of Eahy926 cells and A549 cells (P > 0.1); (B and C) Cell cycle analysis was performed on FACSCalibur flow cytometer. The percentages of cell population in subG 1 , G 1 , S or G 2 /M phases were calculated from histograms by using the CellQuest software; The data represent the mean ± SD of three independent experiments (P > 0.05).

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Cytometry, Software

    9) Product Images from "Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model"

    Article Title: Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00317.x

    Ad.EGFP-transduced MSCs transfer adenoviral-encoded transgene expression onto A549 cells. ( A ) MSC cells were transduced with Ad.EGFP at 10, 100 or 1000 pfu/cell. After 48 hrs MSCs were trypsinized and prepared as previously described. Before addition to A549 seeded wells the Ad.EGFP-transduced MSC cell suspension was centrifuged. The supernatant was removed from the cell pellet and also added to A549 cells (white bars, which are hardly visible except for 1000 pfu). This resulted in almost no transduction, indicating that no virus was left in the supernatant medium or produced in the MSCs. The MSCs themselves were re-suspended in a volume of medium equivalent to that of the aspirated supernatant. 10 4 Ad.EGFP-transduced MSCs (black bars) were added to wells seeded with A549 cells. Samples were assayed for EGFP expression by flow cytometry after a further 48 hrs. ( B ) Adenovirus-transduced MSC cocultured with 293 cells results in novel virus genome production. MSCs were transduced with Ad.BGal (100 pfu/cell) or left untreated. After 48 hrs, untransduced (MSC+293) and Ad-transduced MSCs (MSC Ad.+293) were cocultured with 293 cells that had been seeded the previous day at 10 5 cells/well or Ad.BGal-transduced MSCs were replated into wells containing no 293 cells (MSC Ad.). After 24 hrs, cells were harvested and DNA was extracted. Cells were analysed for viral DNA content by quantitative PCR using primers specific for the knob turn region of the virus and the SYBR Green quantitative PCR system. Samples were run on an ABI Prism 7000 sequence detector. Please note that the graph is depicted in log-scale. ( C ) In order to rule out that our MSCs expressed adenoviral E1A and could facilitate virus replication, we performed a Western blot on protein lysates from MSCs, MSCs transduced with Ad.EGFP (0, 48, 72 and 96 hrs after transduction) and 293 cells as positive control. Only 293 cells showed a signal for E1A. A CuZnSOD Western blot was carried out as loading control.
    Figure Legend Snippet: Ad.EGFP-transduced MSCs transfer adenoviral-encoded transgene expression onto A549 cells. ( A ) MSC cells were transduced with Ad.EGFP at 10, 100 or 1000 pfu/cell. After 48 hrs MSCs were trypsinized and prepared as previously described. Before addition to A549 seeded wells the Ad.EGFP-transduced MSC cell suspension was centrifuged. The supernatant was removed from the cell pellet and also added to A549 cells (white bars, which are hardly visible except for 1000 pfu). This resulted in almost no transduction, indicating that no virus was left in the supernatant medium or produced in the MSCs. The MSCs themselves were re-suspended in a volume of medium equivalent to that of the aspirated supernatant. 10 4 Ad.EGFP-transduced MSCs (black bars) were added to wells seeded with A549 cells. Samples were assayed for EGFP expression by flow cytometry after a further 48 hrs. ( B ) Adenovirus-transduced MSC cocultured with 293 cells results in novel virus genome production. MSCs were transduced with Ad.BGal (100 pfu/cell) or left untreated. After 48 hrs, untransduced (MSC+293) and Ad-transduced MSCs (MSC Ad.+293) were cocultured with 293 cells that had been seeded the previous day at 10 5 cells/well or Ad.BGal-transduced MSCs were replated into wells containing no 293 cells (MSC Ad.). After 24 hrs, cells were harvested and DNA was extracted. Cells were analysed for viral DNA content by quantitative PCR using primers specific for the knob turn region of the virus and the SYBR Green quantitative PCR system. Samples were run on an ABI Prism 7000 sequence detector. Please note that the graph is depicted in log-scale. ( C ) In order to rule out that our MSCs expressed adenoviral E1A and could facilitate virus replication, we performed a Western blot on protein lysates from MSCs, MSCs transduced with Ad.EGFP (0, 48, 72 and 96 hrs after transduction) and 293 cells as positive control. Only 293 cells showed a signal for E1A. A CuZnSOD Western blot was carried out as loading control.

    Techniques Used: Expressing, Transduction, Produced, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, SYBR Green Assay, Sequencing, Western Blot, Positive Control

    Ad.TR-transduced MSCs induce apoptosis in A549 cells in the presence of washed blood (erythrocytes) and white blood cells at physiological concentrations. MSCs were untransduced or transduced with Ad.BGal (100 pfu/cell) or Ad.TR (100 pfu/cell) (MSC, MSCβGal, MSCTR) and harvested after 48 hrs. A549 cells were stained with cell tracker green and seeded into wells at 10 5 cells per well. Once these cells had adhered to the tissue culture wells ( A ) white blood cells were added at a concentration of 3 × 10 6 cells/ml or ( B ) washed blood cells were added at approximately 5 × 10 9 cells/ml. 10 4 untransduced or Ad-transduced MSC cells were added to each well. After 48 hrs, washed blood cells ( A ) or white blood cells ( B ) and medium alone containing wells were harvested and prepared for Nicoletti assay. The wells containing washed blood cells were harvested and the red blood cells were lysed by exposure to a hypotonic solution before these samples were prepared for Nicoletti assay. All samples were assayed by flow cytometry gating on the cell tracker green positive population alone. Overall, the level of apoptosis in white blood cell and washed blood exposed wells was increased, however, samples exposed to Ad.TR-transduced MSCs were consistently higher than background or controls. Numbers represent mean values of three experiments ± standard deviation. * P
    Figure Legend Snippet: Ad.TR-transduced MSCs induce apoptosis in A549 cells in the presence of washed blood (erythrocytes) and white blood cells at physiological concentrations. MSCs were untransduced or transduced with Ad.BGal (100 pfu/cell) or Ad.TR (100 pfu/cell) (MSC, MSCβGal, MSCTR) and harvested after 48 hrs. A549 cells were stained with cell tracker green and seeded into wells at 10 5 cells per well. Once these cells had adhered to the tissue culture wells ( A ) white blood cells were added at a concentration of 3 × 10 6 cells/ml or ( B ) washed blood cells were added at approximately 5 × 10 9 cells/ml. 10 4 untransduced or Ad-transduced MSC cells were added to each well. After 48 hrs, washed blood cells ( A ) or white blood cells ( B ) and medium alone containing wells were harvested and prepared for Nicoletti assay. The wells containing washed blood cells were harvested and the red blood cells were lysed by exposure to a hypotonic solution before these samples were prepared for Nicoletti assay. All samples were assayed by flow cytometry gating on the cell tracker green positive population alone. Overall, the level of apoptosis in white blood cell and washed blood exposed wells was increased, however, samples exposed to Ad.TR-transduced MSCs were consistently higher than background or controls. Numbers represent mean values of three experiments ± standard deviation. * P

    Techniques Used: Transduction, Staining, Concentration Assay, Nicoletti Assay, Flow Cytometry, Cytometry, Standard Deviation

    MSCs transduced with Ad.TR give rise to tumour growth reduction in vivo . ( A ) Immune-deficient mice were injected with 5 × 10 6 A549 cells into the right flank. After 1 week when tumours were approximately 100 mm 3 in size, 1 × 10 6 MSCs either transduced with Ad.TR or Ad.EGFP were directly injected into the tumour. The tumour growth was followed over 3 weeks. This is depicted in the graph. Numbers represent mean values of five animals in each group ± standard deviation. ** P
    Figure Legend Snippet: MSCs transduced with Ad.TR give rise to tumour growth reduction in vivo . ( A ) Immune-deficient mice were injected with 5 × 10 6 A549 cells into the right flank. After 1 week when tumours were approximately 100 mm 3 in size, 1 × 10 6 MSCs either transduced with Ad.TR or Ad.EGFP were directly injected into the tumour. The tumour growth was followed over 3 weeks. This is depicted in the graph. Numbers represent mean values of five animals in each group ± standard deviation. ** P

    Techniques Used: Transduction, In Vivo, Mouse Assay, Injection, Standard Deviation

    Comparison of neutralization properties of different human sera. ( A ) 10 7 Ad.EGFP pfu were incubated in complete RPMI medium (white bar) or human serum (black bars) diluted 1/10 or 1/100 in complete RPMI medium for 30 min. at 37°C. Data shown are samples with high (Serum A) and low (Serum B) adenovirus-neutralizing activities chosen from a panel of 18 donors. After pre-incubation of Ad.EGFP with medium and the two sera, the samples were added to wells containing A549 cells seeded the previous day at 10 5 cells per well. After 48 hrs, the cells were harvested and analysed for EGFP expression by flow cytometry. While A549 cells are readily transduced with Ad.EGFP pre-incubated in RPMI medium (white bar), pre-incubation with Serum A at both concentrations completely neutralizes the transduction activity of the adenoviral vector. Serum B, on the other side allows transduction of A549 cells after pre-incubation Numbers represent mean values of duplicate experiments. ( B ) Exposing MSCs transduced with Ad.EGFP to neat human serum with high levels of adenovirus neutralizing antibodies (Serum A) does not inhibit the ability of these cells to ‘post-transduce’ A549 cells. MSC cells were transduced with Ad.EGFP (100 pfu/cell). After 48 hrs, the cells were harvested and incubated in neat serum or serum diluted 1:10 or 1:100 in complete DMEM medium for 30 min. at 37°C. Samples were prepared using serum from a high neutralizing donor (Serum A; white bars) and a low-neutralizing donor (Serum B; black bars). 10 4 MSCs including the serum were then added to wells containing A549 cells seeded the previous day. After 48 hrs, samples were prepared for analysis by flow cytometry. This analysis shows that A549 cells were transduced by the Ad.EGFP-loaded MSCs regardless of the concentration of adenovirus-neutralizing antibodies. Numbers represent mean values of three samples ± standard deviation. ( C ) Exposing MSCs transduced with Ad.TR to neat human serum with high levels of adenovirus neutralizing antibodies does not inhibit the ability of these cells to induce apoptosis in A549 cells. MSC cells were untransduced (MSC) or transduced with Ad.BGal (MSCβGal) or Ad.TR (MSCTR) with 100 pfu/cell. A549 cells that were treated with serum only are shown on the left (serum). After 24 hrs, the cells were harvested and incubated in neat serum for 30 min. at 37 C. The cell suspension (10 4 MSC cells/well) and serum were then added to wells containing A549 cells seeded the previous day. After 48 hrs, samples were prepared for Nicoletti assay and analysed by flow cytometry. Numbers represent mean values of duplicate samples ± standard deviation. * P
    Figure Legend Snippet: Comparison of neutralization properties of different human sera. ( A ) 10 7 Ad.EGFP pfu were incubated in complete RPMI medium (white bar) or human serum (black bars) diluted 1/10 or 1/100 in complete RPMI medium for 30 min. at 37°C. Data shown are samples with high (Serum A) and low (Serum B) adenovirus-neutralizing activities chosen from a panel of 18 donors. After pre-incubation of Ad.EGFP with medium and the two sera, the samples were added to wells containing A549 cells seeded the previous day at 10 5 cells per well. After 48 hrs, the cells were harvested and analysed for EGFP expression by flow cytometry. While A549 cells are readily transduced with Ad.EGFP pre-incubated in RPMI medium (white bar), pre-incubation with Serum A at both concentrations completely neutralizes the transduction activity of the adenoviral vector. Serum B, on the other side allows transduction of A549 cells after pre-incubation Numbers represent mean values of duplicate experiments. ( B ) Exposing MSCs transduced with Ad.EGFP to neat human serum with high levels of adenovirus neutralizing antibodies (Serum A) does not inhibit the ability of these cells to ‘post-transduce’ A549 cells. MSC cells were transduced with Ad.EGFP (100 pfu/cell). After 48 hrs, the cells were harvested and incubated in neat serum or serum diluted 1:10 or 1:100 in complete DMEM medium for 30 min. at 37°C. Samples were prepared using serum from a high neutralizing donor (Serum A; white bars) and a low-neutralizing donor (Serum B; black bars). 10 4 MSCs including the serum were then added to wells containing A549 cells seeded the previous day. After 48 hrs, samples were prepared for analysis by flow cytometry. This analysis shows that A549 cells were transduced by the Ad.EGFP-loaded MSCs regardless of the concentration of adenovirus-neutralizing antibodies. Numbers represent mean values of three samples ± standard deviation. ( C ) Exposing MSCs transduced with Ad.TR to neat human serum with high levels of adenovirus neutralizing antibodies does not inhibit the ability of these cells to induce apoptosis in A549 cells. MSC cells were untransduced (MSC) or transduced with Ad.BGal (MSCβGal) or Ad.TR (MSCTR) with 100 pfu/cell. A549 cells that were treated with serum only are shown on the left (serum). After 24 hrs, the cells were harvested and incubated in neat serum for 30 min. at 37 C. The cell suspension (10 4 MSC cells/well) and serum were then added to wells containing A549 cells seeded the previous day. After 48 hrs, samples were prepared for Nicoletti assay and analysed by flow cytometry. Numbers represent mean values of duplicate samples ± standard deviation. * P

    Techniques Used: Neutralization, Incubation, Expressing, Flow Cytometry, Cytometry, Transduction, Activity Assay, Plasmid Preparation, Concentration Assay, Standard Deviation, Nicoletti Assay

    MSCs transduced with Ad.TR can induce apoptosis in A549 cells. ( A ) MSCs were transduced with Ad.BGal (100 pfu/cell) or Ad.TR (100 pfu/cell). After 48 hrs MSCs were trypsinized and 10 4 of these cells were added to wells that had been seeded the day before with A549 cells at 10 5 cells per well. After 48 hrs, wells were trypsinized and the complete mixed cell population was measured by Nicoletti apoptosis assay. A549 cells and MSCs cultured alone as well as A549 cells mixed with MSCs transduced with Ad.BGal showed only background apoptosis of below 5%. In contrast, in A549 mixed with MSCs transduced with Ad.TR apoptosis rates of almost 30% could be measured. This apoptosis could be inhibited by TRAIL neutralizing antibodies (α-TR-ab) and the pan-caspase inhibitor zVAD. Numbers represent mean values of three samples ± standard deviation. ** P
    Figure Legend Snippet: MSCs transduced with Ad.TR can induce apoptosis in A549 cells. ( A ) MSCs were transduced with Ad.BGal (100 pfu/cell) or Ad.TR (100 pfu/cell). After 48 hrs MSCs were trypsinized and 10 4 of these cells were added to wells that had been seeded the day before with A549 cells at 10 5 cells per well. After 48 hrs, wells were trypsinized and the complete mixed cell population was measured by Nicoletti apoptosis assay. A549 cells and MSCs cultured alone as well as A549 cells mixed with MSCs transduced with Ad.BGal showed only background apoptosis of below 5%. In contrast, in A549 mixed with MSCs transduced with Ad.TR apoptosis rates of almost 30% could be measured. This apoptosis could be inhibited by TRAIL neutralizing antibodies (α-TR-ab) and the pan-caspase inhibitor zVAD. Numbers represent mean values of three samples ± standard deviation. ** P

    Techniques Used: Transduction, Apoptosis Assay, Cell Culture, Standard Deviation

    Adenovirally expressed TRAIL induces higher levels of apoptosis than recombinant TRAIL in A549 cells. ( A ) Replication-defective adenoviral vectors with deletions of the E1 and E3 regions (Ad. ΔE1 and Ad. ΔE3) were used in our study. They expressed TRAIL (Ad.TR), β-galactosidase (Ad.BGal) or EGFP (Ad.EGFP) from the cytomegalovirus (CMV) promoter (P-CMV). Other elements are the 5′ and 3′ inverted-terminal-repeats (ITR) and the thymidine kinase polyA-addition signal (TKpA). ( B ) A549 cells were seeded at 10 5 cells per well overnight. Adenovirus (expressing TRAIL or β-galactosidase, Ad.TR and Ad.BGal, respectively) was added at 1, 10 or 100 pfu/cell or recombinant TRAIL (rTR) was added at 1, 5 or 10 ng/ml. As positive control for the effects of rTR human colorectal cancer HCT116 cells were treated with 10 ng/ml rTR. Cells were assayed for apoptosis after 48 hrs by Nicoletti hypodiploidity assay. Samples were analysed by flow cytometry. Numbers represent mean values of five samples ± standard deviation. ** P
    Figure Legend Snippet: Adenovirally expressed TRAIL induces higher levels of apoptosis than recombinant TRAIL in A549 cells. ( A ) Replication-defective adenoviral vectors with deletions of the E1 and E3 regions (Ad. ΔE1 and Ad. ΔE3) were used in our study. They expressed TRAIL (Ad.TR), β-galactosidase (Ad.BGal) or EGFP (Ad.EGFP) from the cytomegalovirus (CMV) promoter (P-CMV). Other elements are the 5′ and 3′ inverted-terminal-repeats (ITR) and the thymidine kinase polyA-addition signal (TKpA). ( B ) A549 cells were seeded at 10 5 cells per well overnight. Adenovirus (expressing TRAIL or β-galactosidase, Ad.TR and Ad.BGal, respectively) was added at 1, 10 or 100 pfu/cell or recombinant TRAIL (rTR) was added at 1, 5 or 10 ng/ml. As positive control for the effects of rTR human colorectal cancer HCT116 cells were treated with 10 ng/ml rTR. Cells were assayed for apoptosis after 48 hrs by Nicoletti hypodiploidity assay. Samples were analysed by flow cytometry. Numbers represent mean values of five samples ± standard deviation. ** P

    Techniques Used: Recombinant, Expressing, Positive Control, Flow Cytometry, Cytometry, Standard Deviation

    10) Product Images from "Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin"

    Article Title: Modulation of NF-κB/miR-21/PTEN Pathway Sensitizes Non-Small Cell Lung Cancer to Cisplatin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121547

    miR-21 modulated PTEN expression and cell viability in A549 cells. The cells in control, miR-21 mimics and miR-21 inhibitor were transfected with 100 nM scrambled oligonucleotide, miR-21 mimic and miR-21 inhibitor, respectively. (A) Time axis of the experiment. The scrambled oligonucleotide, miR-21 mimic or inhibitor was given 4h before cisplatin treatment (5 μM). And 48h after cisplatin treatment, the cells were harvested for further analysis. (B) The mRNA expression of PTEN was inversely related with miR-21. (C) miR-21 mimics increased the cell viability while miR-21 inhibitor decreased it when cells exposed to cisplatin treatment. (D)Western blotting of PTEN, total Akt (t-Akt) and phosphor ser473 Akt. (E) Graphic representation of PTEN, t-Akt and phosphor-Akt. miR-21 decreased the expression of PTEN and increased the ratio of phosphor-Akt. * compared with control group, # compared with miR-21 mimic group. P
    Figure Legend Snippet: miR-21 modulated PTEN expression and cell viability in A549 cells. The cells in control, miR-21 mimics and miR-21 inhibitor were transfected with 100 nM scrambled oligonucleotide, miR-21 mimic and miR-21 inhibitor, respectively. (A) Time axis of the experiment. The scrambled oligonucleotide, miR-21 mimic or inhibitor was given 4h before cisplatin treatment (5 μM). And 48h after cisplatin treatment, the cells were harvested for further analysis. (B) The mRNA expression of PTEN was inversely related with miR-21. (C) miR-21 mimics increased the cell viability while miR-21 inhibitor decreased it when cells exposed to cisplatin treatment. (D)Western blotting of PTEN, total Akt (t-Akt) and phosphor ser473 Akt. (E) Graphic representation of PTEN, t-Akt and phosphor-Akt. miR-21 decreased the expression of PTEN and increased the ratio of phosphor-Akt. * compared with control group, # compared with miR-21 mimic group. P

    Techniques Used: Expressing, Transfection

    NF-κB targeting its binding elements in the miR-21 gene promoter. (A) The full length of miR-21 has four NF-κB binding elements. Different lengths of miR-21 promoter were cloned to pGL4 vector. The four elements were mutated individually and the mutated promoters were also cloned to pGL4 vector. (B) The relative luciferase activity in A549 cells cotransfected with pcNF-κB and pGL4 reporting vector. (C) ChIP realtime assay showed that NF-κB antibody bind more DNA than normal IgG. Primer 1 to 4 means the primer contained the four NF-κB binding elements from upstream to downstream. * P
    Figure Legend Snippet: NF-κB targeting its binding elements in the miR-21 gene promoter. (A) The full length of miR-21 has four NF-κB binding elements. Different lengths of miR-21 promoter were cloned to pGL4 vector. The four elements were mutated individually and the mutated promoters were also cloned to pGL4 vector. (B) The relative luciferase activity in A549 cells cotransfected with pcNF-κB and pGL4 reporting vector. (C) ChIP realtime assay showed that NF-κB antibody bind more DNA than normal IgG. Primer 1 to 4 means the primer contained the four NF-κB binding elements from upstream to downstream. * P

    Techniques Used: Binding Assay, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation

    NF-κB/miR-21/PTEN pathway modulated cell viability. (A) mRNA expressions of NF-κB, miR-21 and PTEN in the cells transduced with Lenti-control, Lenti-NF-κB and Lenti-shNF-κB. The miR-21 level was significantly increased while the PTEN was decreased in the cells transduced with Lenti-NF-κB compared with control vector. The miR-21 level was significantly decreased while the PTEN was decreased in the cells transduced with Lenti-NF-κB. (B) Cell viability was increased by Lenti-NF-κB while decreased by Lenti-shNF-κB. (C) Overexpression of PTEN restored the sensitivity of A549 cells to cisplatin treatment compared with overexpression of NF-κB or miR-21. * compared with control group, # compared with Lenti-NF-κB group. P
    Figure Legend Snippet: NF-κB/miR-21/PTEN pathway modulated cell viability. (A) mRNA expressions of NF-κB, miR-21 and PTEN in the cells transduced with Lenti-control, Lenti-NF-κB and Lenti-shNF-κB. The miR-21 level was significantly increased while the PTEN was decreased in the cells transduced with Lenti-NF-κB compared with control vector. The miR-21 level was significantly decreased while the PTEN was decreased in the cells transduced with Lenti-NF-κB. (B) Cell viability was increased by Lenti-NF-κB while decreased by Lenti-shNF-κB. (C) Overexpression of PTEN restored the sensitivity of A549 cells to cisplatin treatment compared with overexpression of NF-κB or miR-21. * compared with control group, # compared with Lenti-NF-κB group. P

    Techniques Used: Transduction, Plasmid Preparation, Over Expression

    11) Product Images from "A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication"

    Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124651

    Lentivirus-delivered FGFR1 overexpression affected influenza A virus internalization, but not binding, during virus entry. A549 cells with FGFR1, FGFR4, or GFP overexpression were pretreated with or without sialidase, and then infected with influenza A/PR8 ( A ) or H5N1 ( B ). The procedures were described in detail in the Methods, virus binding and internalization assay. Influenza virus NP was detected by Western blotting using anti-influenza NP antibodies. β-actin protein was used as an internal control. The ratio of NP/β-actin was determined based on densitometric analysis. The results were expressed as means ± s.e.m. (n = 3). Values of P
    Figure Legend Snippet: Lentivirus-delivered FGFR1 overexpression affected influenza A virus internalization, but not binding, during virus entry. A549 cells with FGFR1, FGFR4, or GFP overexpression were pretreated with or without sialidase, and then infected with influenza A/PR8 ( A ) or H5N1 ( B ). The procedures were described in detail in the Methods, virus binding and internalization assay. Influenza virus NP was detected by Western blotting using anti-influenza NP antibodies. β-actin protein was used as an internal control. The ratio of NP/β-actin was determined based on densitometric analysis. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Techniques Used: Over Expression, Binding Assay, Infection, Western Blot

    Time-course analysis of FGFR family member expression in A549 cells infected with influenza A/PR/8/34 virus. ( A - D ) A549 cells were infected with PR8 virus at an MOI of 1 for the indicated times. FGFR mRNA levels were detected using real-time PCR analysis. The mRNA expression of FGFR1 ( A ), FGFR2 ( B ), FGFR3 ( C ), and FGFR4 ( D ) relative to the reference gene GAPDH was calculated. ( E , F ) The lysates of A549 cells were obtained at the indicated times post-PR8 infection. Protein levels of FGFR1 ( E ) and FGFR4 ( F ) were determined by Western blotting using specific antibodies. Densitometric analysis relative to β-actin levels was expressed as fold change. All graphs present the means ± s.e.m. (n = 3). Values of P
    Figure Legend Snippet: Time-course analysis of FGFR family member expression in A549 cells infected with influenza A/PR/8/34 virus. ( A - D ) A549 cells were infected with PR8 virus at an MOI of 1 for the indicated times. FGFR mRNA levels were detected using real-time PCR analysis. The mRNA expression of FGFR1 ( A ), FGFR2 ( B ), FGFR3 ( C ), and FGFR4 ( D ) relative to the reference gene GAPDH was calculated. ( E , F ) The lysates of A549 cells were obtained at the indicated times post-PR8 infection. Protein levels of FGFR1 ( E ) and FGFR4 ( F ) were determined by Western blotting using specific antibodies. Densitometric analysis relative to β-actin levels was expressed as fold change. All graphs present the means ± s.e.m. (n = 3). Values of P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Western Blot

    Lentivirus-mediated FGFR1 overexpression in A549 cells significantly decreased influenza A virus replication. A549 cells were infected with recombinant lentivirus expressing FGFR1, FGFR4, or GFP (as a control). After 48 h, A549 cells were infected with PR8 virus (MOI = 1). The supernatants and lysates of A549 cells were harvested after 24 h virus infection. ( A , B ) Influenza M1 mRNA levels in A549 cells with PR8 and H5N1 were detected using real-time PCR. ( C , D ) Progeny virus titers of PR8 and H5N1 were determined as described previously. ( E , F ) FGFR1 and FGFR4 expression efficiencies were detected using real-time PCR and Western blotting. All graphs present the means ± s.e.m. (n = 3). Values of P
    Figure Legend Snippet: Lentivirus-mediated FGFR1 overexpression in A549 cells significantly decreased influenza A virus replication. A549 cells were infected with recombinant lentivirus expressing FGFR1, FGFR4, or GFP (as a control). After 48 h, A549 cells were infected with PR8 virus (MOI = 1). The supernatants and lysates of A549 cells were harvested after 24 h virus infection. ( A , B ) Influenza M1 mRNA levels in A549 cells with PR8 and H5N1 were detected using real-time PCR. ( C , D ) Progeny virus titers of PR8 and H5N1 were determined as described previously. ( E , F ) FGFR1 and FGFR4 expression efficiencies were detected using real-time PCR and Western blotting. All graphs present the means ± s.e.m. (n = 3). Values of P

    Techniques Used: Over Expression, Infection, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Repression of FGFR1 phosphorylation by the PD173074 inhibitor enhanced influenza A/PR8virus replication. (A) The lysates of A549 cells with PR8 virus infection were harvested for the indicated times. FGFR1 phosphorylation levels were determined by Western blotting with phospho-specific antibodies. Densitometric analysis of the phosphorylated/total FGFR1 ratio was shown as fold change. (B-D) A549 cells were pretreated with PD173074 (FGFR1 kinase inhibitor) at indicated concentrations for 30 min, and then infected with PR8 (indicated-dose inhibitor was also added to virus diluent). After 1 h, the medium was replaced with fresh medium supplemented with PD173074. (B) The cell lysates were collected at 12 hpi and then subjected to Western blotting for FGFR1 phosphorylation detection. The phosphorylated/total FGFR1 ratio indicated the fold change of FGFR1 phosphorylation. (C) Influenza M1 mRNA in PR8 infected-A549 cells with PD173074 treatment as indicated was detected by real-time PCR. (D) MTT assay of A549 cells with PD173074 treatment as indicated with or without PR8 infection. The results were expressed as means ± s.e.m. (n = 3). Values of P
    Figure Legend Snippet: Repression of FGFR1 phosphorylation by the PD173074 inhibitor enhanced influenza A/PR8virus replication. (A) The lysates of A549 cells with PR8 virus infection were harvested for the indicated times. FGFR1 phosphorylation levels were determined by Western blotting with phospho-specific antibodies. Densitometric analysis of the phosphorylated/total FGFR1 ratio was shown as fold change. (B-D) A549 cells were pretreated with PD173074 (FGFR1 kinase inhibitor) at indicated concentrations for 30 min, and then infected with PR8 (indicated-dose inhibitor was also added to virus diluent). After 1 h, the medium was replaced with fresh medium supplemented with PD173074. (B) The cell lysates were collected at 12 hpi and then subjected to Western blotting for FGFR1 phosphorylation detection. The phosphorylated/total FGFR1 ratio indicated the fold change of FGFR1 phosphorylation. (C) Influenza M1 mRNA in PR8 infected-A549 cells with PD173074 treatment as indicated was detected by real-time PCR. (D) MTT assay of A549 cells with PD173074 treatment as indicated with or without PR8 infection. The results were expressed as means ± s.e.m. (n = 3). Values of P

    Techniques Used: Infection, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay

    Specific siRNA target FGFR1 markedly increased PR8 virus entry at an early stage of the viral life cycle. ( A - C ) A549 cells were transfected with FGFR1 siRNA#1, FGFR4 siRNA#1, and negative control siRNA. After 48 h of transduction, A549 cells were incubated with PR8 virus at an MOI of 0.01 for 4 h, followed by indirect immunofluorescence assays. A549 cells were stained with anti-influenza A virus NP antibodies (green) and Hoechst 33342 (nucleus, blue). ( D ) The data of PR8-infected cells were presented as the percentages of NP-positive cells to the total number of cells. The bars represent the means ± s.e.m. (n = 3). P
    Figure Legend Snippet: Specific siRNA target FGFR1 markedly increased PR8 virus entry at an early stage of the viral life cycle. ( A - C ) A549 cells were transfected with FGFR1 siRNA#1, FGFR4 siRNA#1, and negative control siRNA. After 48 h of transduction, A549 cells were incubated with PR8 virus at an MOI of 0.01 for 4 h, followed by indirect immunofluorescence assays. A549 cells were stained with anti-influenza A virus NP antibodies (green) and Hoechst 33342 (nucleus, blue). ( D ) The data of PR8-infected cells were presented as the percentages of NP-positive cells to the total number of cells. The bars represent the means ± s.e.m. (n = 3). P

    Techniques Used: Transfection, Negative Control, Transduction, Incubation, Immunofluorescence, Staining, Infection

    FGFR1 silencing by RNAi increased influenza A/PR8 and H5N1 virus replication. ( A - D ) A549 cells were transiently transfected with specific siRNA targeting FGFR1, FGFR4, or negative control siRNA. Forty-eight hours later, A549 cells were infected with PR8 virus at an MOI of 1. The cell culture supernatants and cell lysates were obtained at 24 hpi. Influenza virus M1 mRNA expression in A549 cells with PR8 ( A ) or H5N1 ( B ) infection was detected using real-time PCR. Progeny virus titers of PR8 ( C ) or H5N1 ( D ) were determined using MDCK cells with the TCID 50 assay. ( E ) The knockdown efficiencies of FGFR1 and FGFR4 by target siRNA were tested using real-time PCR. ( F ) Protein expressions of FGFR1 and FGFR4 were detected using specific antibodies by Western blotting assay. All graphs represent the means ± s.e.m. (n = 3). Values of P
    Figure Legend Snippet: FGFR1 silencing by RNAi increased influenza A/PR8 and H5N1 virus replication. ( A - D ) A549 cells were transiently transfected with specific siRNA targeting FGFR1, FGFR4, or negative control siRNA. Forty-eight hours later, A549 cells were infected with PR8 virus at an MOI of 1. The cell culture supernatants and cell lysates were obtained at 24 hpi. Influenza virus M1 mRNA expression in A549 cells with PR8 ( A ) or H5N1 ( B ) infection was detected using real-time PCR. Progeny virus titers of PR8 ( C ) or H5N1 ( D ) were determined using MDCK cells with the TCID 50 assay. ( E ) The knockdown efficiencies of FGFR1 and FGFR4 by target siRNA were tested using real-time PCR. ( F ) Protein expressions of FGFR1 and FGFR4 were detected using specific antibodies by Western blotting assay. All graphs represent the means ± s.e.m. (n = 3). Values of P

    Techniques Used: Transfection, Negative Control, Infection, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    12) Product Images from "Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development"

    Article Title: Porous Polymeric Microspheres With Controllable Pore Diameters for Tissue Engineered Lung Tumor Model Development

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.00799

    In vitro drug screening studies. Comparative in vitro screening of anti-cancer drugs on A549-MRC-5 monolayer and col-PPMS co-culture treatment groups after 72 h of treatment showed significant difference in cell viability between both cultures. The col-PPMS-based cultures showing significant higher resistance to the drugs than monolayer cultures. Significance was analyzed by Student’s t- test (* p
    Figure Legend Snippet: In vitro drug screening studies. Comparative in vitro screening of anti-cancer drugs on A549-MRC-5 monolayer and col-PPMS co-culture treatment groups after 72 h of treatment showed significant difference in cell viability between both cultures. The col-PPMS-based cultures showing significant higher resistance to the drugs than monolayer cultures. Significance was analyzed by Student’s t- test (* p

    Techniques Used: In Vitro, Co-Culture Assay

    Fluorescence Live/Dead imaging of monolayered and col-PPMS-cocultured A549 and MRC-5 after 72 h of drug treatment showing greater viability (green) for col-PPMS co-cultures than for the monolayer co-cultures in almost all cases. For the monolayer co-cultures, most cells were either washed away or floating due to cell death, following treatment. Insert images are phase contrast images of the same view. Scale bar: 400 μm.
    Figure Legend Snippet: Fluorescence Live/Dead imaging of monolayered and col-PPMS-cocultured A549 and MRC-5 after 72 h of drug treatment showing greater viability (green) for col-PPMS co-cultures than for the monolayer co-cultures in almost all cases. For the monolayer co-cultures, most cells were either washed away or floating due to cell death, following treatment. Insert images are phase contrast images of the same view. Scale bar: 400 μm.

    Techniques Used: Fluorescence, Imaging

    Cell seeding optimization experiments. PPMS facilitated greater A549 cell attachment and growth compared to PLGAMS. Seeding densities used include (A) 5 × 10 3 , (B) 10 × 10 3 , (C) 25 × 10 3 , (D) 50 × 10 3 cells/mg of microparticles/well. Data as mean ± SD, n = 3; (E) Live/Dead staining shows greater cell death (red) on PLGAMS than on PPMS, which had more viable cells (green) (optimized cell seeding density – 25 × 10 3 cells/mg/well; scale bars = 100 μm). Significant difference was analyzed by Student’s t -test (* p
    Figure Legend Snippet: Cell seeding optimization experiments. PPMS facilitated greater A549 cell attachment and growth compared to PLGAMS. Seeding densities used include (A) 5 × 10 3 , (B) 10 × 10 3 , (C) 25 × 10 3 , (D) 50 × 10 3 cells/mg of microparticles/well. Data as mean ± SD, n = 3; (E) Live/Dead staining shows greater cell death (red) on PLGAMS than on PPMS, which had more viable cells (green) (optimized cell seeding density – 25 × 10 3 cells/mg/well; scale bars = 100 μm). Significant difference was analyzed by Student’s t -test (* p

    Techniques Used: Cell Attachment Assay, Staining

    Co-culture of MRC-5 and A549 (1:1 seeding ratio) at (A) low magnification (scale bar: 100 μm) and (B) high magnification (scale bar: 20 μm). Left, phase contrast images; right, overlay of green (FITC channel) and red (Cy5 channel) fluorescent images. (C) Quantification of number of cells attached onto Col-PPMS and PPMS showing significantly higher cell attachment on Col-PPMS at almost all timepoints. * p
    Figure Legend Snippet: Co-culture of MRC-5 and A549 (1:1 seeding ratio) at (A) low magnification (scale bar: 100 μm) and (B) high magnification (scale bar: 20 μm). Left, phase contrast images; right, overlay of green (FITC channel) and red (Cy5 channel) fluorescent images. (C) Quantification of number of cells attached onto Col-PPMS and PPMS showing significantly higher cell attachment on Col-PPMS at almost all timepoints. * p

    Techniques Used: Co-Culture Assay, Cell Attachment Assay

    13) Product Images from "Overexpression of LRRC59 Is Associated with Poor Prognosis and Promotes Cell Proliferation and Invasion in Lung Adenocarcinoma"

    Article Title: Overexpression of LRRC59 Is Associated with Poor Prognosis and Promotes Cell Proliferation and Invasion in Lung Adenocarcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S245336

    Knockdown LRRC59 impaired the migratory and invasive abilities of lung cancer cells. ( A, B ) Transwell migration analysis of shCon and shLRRC59 lung cancer cells. Representative images of migration assays in A549 and H1299 cells (magnification, 200×) are shown. And histograms of migration results were shown in ( B ). Data were representative results from experiments repeated at least three times (***P
    Figure Legend Snippet: Knockdown LRRC59 impaired the migratory and invasive abilities of lung cancer cells. ( A, B ) Transwell migration analysis of shCon and shLRRC59 lung cancer cells. Representative images of migration assays in A549 and H1299 cells (magnification, 200×) are shown. And histograms of migration results were shown in ( B ). Data were representative results from experiments repeated at least three times (***P

    Techniques Used: Migration

    Knockdown LRRC59 induced the apoptosis of A549 cells, but not p53 -deficient H1299 cells. Apoptosis in shCon and shLRRC59 lung cancer cells were collected for Annexin V and PI staining, followed by flow cytometry analysis. Abbreviations: PE-A, phycoerythrin-A; FITC-A, fluorescein isothiocyanate-A; PI, propidium iodide; shCON, short hairpin RNA-control; sh, short hairpin RNA.
    Figure Legend Snippet: Knockdown LRRC59 induced the apoptosis of A549 cells, but not p53 -deficient H1299 cells. Apoptosis in shCon and shLRRC59 lung cancer cells were collected for Annexin V and PI staining, followed by flow cytometry analysis. Abbreviations: PE-A, phycoerythrin-A; FITC-A, fluorescein isothiocyanate-A; PI, propidium iodide; shCON, short hairpin RNA-control; sh, short hairpin RNA.

    Techniques Used: Staining, Flow Cytometry, shRNA

    14) Product Images from "A novel lncRNA, LUADT1, promotes lung adenocarcinoma proliferation via the epigenetic suppression of p27"

    Article Title: A novel lncRNA, LUADT1, promotes lung adenocarcinoma proliferation via the epigenetic suppression of p27

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.203

    The silencing of LUADT1 inhibited LUAD growth in vivo . LUADT1-targeting or scrambled shRNA was transfected into A549 cells, and then, the cells were injected into nude mice. ( a ) LUADT1 expression was downregulated in the shLUADT1-transfected cell-derived xenograft tumors. The xenograft tumor weight ( c ) and volume ( b and d ) in the shLUADT1 group were significantly lower than those in the scrambled shRNA group. IHC staining was performed on xenograft tumors, and the Ki67 staining signal was weaker in the shLUADT1 group than in the scrambled shRNA group ( e ). * P
    Figure Legend Snippet: The silencing of LUADT1 inhibited LUAD growth in vivo . LUADT1-targeting or scrambled shRNA was transfected into A549 cells, and then, the cells were injected into nude mice. ( a ) LUADT1 expression was downregulated in the shLUADT1-transfected cell-derived xenograft tumors. The xenograft tumor weight ( c ) and volume ( b and d ) in the shLUADT1 group were significantly lower than those in the scrambled shRNA group. IHC staining was performed on xenograft tumors, and the Ki67 staining signal was weaker in the shLUADT1 group than in the scrambled shRNA group ( e ). * P

    Techniques Used: In Vivo, shRNA, Transfection, Injection, Mouse Assay, Expressing, Derivative Assay, Immunohistochemistry, Staining

    The silencing of LUADT1 inhibited LUAD cell proliferation in vitro . ( a ) Targeted siRNAs efficiently silenced LUADT1 in the A549 and H1975 cell lines. LUADT1 knockdown inhibited cell proliferation (CCK-8 assay, b ) and clone formation (c ) ability and induced cell cycle arrest at the G0–G1 stage ( d ) in A549 and H1975 cells. BrdU assay showed that siRNA-LUADT1 treatment significantly decreased relative absorbance at 450 nm ( e ). Western blot showed that cyclin D1, CDK4 and CDK6 were decreased after LUADT1 silence in A549 and H1975 cells ( f ). * P
    Figure Legend Snippet: The silencing of LUADT1 inhibited LUAD cell proliferation in vitro . ( a ) Targeted siRNAs efficiently silenced LUADT1 in the A549 and H1975 cell lines. LUADT1 knockdown inhibited cell proliferation (CCK-8 assay, b ) and clone formation (c ) ability and induced cell cycle arrest at the G0–G1 stage ( d ) in A549 and H1975 cells. BrdU assay showed that siRNA-LUADT1 treatment significantly decreased relative absorbance at 450 nm ( e ). Western blot showed that cyclin D1, CDK4 and CDK6 were decreased after LUADT1 silence in A549 and H1975 cells ( f ). * P

    Techniques Used: In Vitro, CCK-8 Assay, BrdU Staining, Western Blot

    15) Product Images from "Chemical Composition and In Vitro Cytotoxicity of Essential Oils from Leaves and Flowers of Callistemon citrinus from Western Himalayas"

    Article Title: Chemical Composition and In Vitro Cytotoxicity of Essential Oils from Leaves and Flowers of Callistemon citrinus from Western Himalayas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133823

    Microscopic images at 10X of A549 (A-F) and C-6 (G-J) cells treated for 24–48 h with 100 μg/mL of leaf and flower oils of C. citrinus (A) Control, no treatment after 24 h. (B) Control, no treatment after 48 h. (C) After 24 h treatment with flower oil. (D) After 48 h treatment with flower oil. (E) After 24 h treatment leaf oil. (F) After 48 h treatment with leaf oil. (G) Control, no treatment after 24 h. (H) Control, no treatment after 48 h. (I) After 24 h treatment with flower oil. (J) After 48 h of treatment with flower oil.
    Figure Legend Snippet: Microscopic images at 10X of A549 (A-F) and C-6 (G-J) cells treated for 24–48 h with 100 μg/mL of leaf and flower oils of C. citrinus (A) Control, no treatment after 24 h. (B) Control, no treatment after 48 h. (C) After 24 h treatment with flower oil. (D) After 48 h treatment with flower oil. (E) After 24 h treatment leaf oil. (F) After 48 h treatment with leaf oil. (G) Control, no treatment after 24 h. (H) Control, no treatment after 48 h. (I) After 24 h treatment with flower oil. (J) After 48 h of treatment with flower oil.

    Techniques Used:

    Effect of leaf and flower oils on cell cycle against A549 (A-F) and C-6 (G-I) cells after 24 h. (A) (D) Control, no treatment. (B) Treated with flower oil at 20 μg/mL. (C) Treated with flower oil at 50 μg/mL. (E) Treated with leaf oil at 50 μg/mL. (F) Treated with leaf oil at 100 μg/mL. (G) Control, no treatment. (H) Treated with flower oil at 50 μg/mL. (I) Treated with flower oil at 100 μg/mL.
    Figure Legend Snippet: Effect of leaf and flower oils on cell cycle against A549 (A-F) and C-6 (G-I) cells after 24 h. (A) (D) Control, no treatment. (B) Treated with flower oil at 20 μg/mL. (C) Treated with flower oil at 50 μg/mL. (E) Treated with leaf oil at 50 μg/mL. (F) Treated with leaf oil at 100 μg/mL. (G) Control, no treatment. (H) Treated with flower oil at 50 μg/mL. (I) Treated with flower oil at 100 μg/mL.

    Techniques Used:

    In vitro cytotoxicity of C . citrinus oil against A549, C-6, Colo-205 and SiHa cells by SRB assay. (A) Flower oil. (B) Leaf oil.
    Figure Legend Snippet: In vitro cytotoxicity of C . citrinus oil against A549, C-6, Colo-205 and SiHa cells by SRB assay. (A) Flower oil. (B) Leaf oil.

    Techniques Used: In Vitro, Sulforhodamine B Assay

    Induction of apoptosis on A549 and C-6 cells by leaf and flower oils at 20, 50 and 100 μg/mL assessed by flow cytometry. (A) A549 cells treated with flower oil for 12 h. (B) A549 cells treated with leaf oils for 12 h. (C) C-6 cells treated with flowers oil for 24 h.
    Figure Legend Snippet: Induction of apoptosis on A549 and C-6 cells by leaf and flower oils at 20, 50 and 100 μg/mL assessed by flow cytometry. (A) A549 cells treated with flower oil for 12 h. (B) A549 cells treated with leaf oils for 12 h. (C) C-6 cells treated with flowers oil for 24 h.

    Techniques Used: Flow Cytometry, Cytometry

    16) Product Images from "The Eukaryotic Elongation Factor 1A Is Critical for Genome Replication of the Paramyxovirus Respiratory Syncytial Virus"

    Article Title: The Eukaryotic Elongation Factor 1A Is Critical for Genome Replication of the Paramyxovirus Respiratory Syncytial Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114447

    Nucleocapsid (N) and phosphoprotein (P) co-localise with host eEF1A in a live virus infection. A549 cells were infected with RSV at a MOI of 1 pfu/cell and fixed for proximity assay 24 h post-infection. Antibodies to eEF1A or eIF3A (negative control), and RSV N and P were used in conjunction with Duolink II PLA probes to detect significant proximity between RSV N, P and eEF1A, and no significant proximity between RSV N, P and eIF3A. ( A ) Images were captured on a confocal microscope and ( B ) the number of signals (foci) detected in 100 cells per reaction was quantified using the Duolink Imagetool software (Olink Biosciences). Data was collected by two readers and the mean number of signals/cell was then calculated. Experiments were repeated three times. Mean values, with SEM. Significance identified using 2-way ANOVA. **P
    Figure Legend Snippet: Nucleocapsid (N) and phosphoprotein (P) co-localise with host eEF1A in a live virus infection. A549 cells were infected with RSV at a MOI of 1 pfu/cell and fixed for proximity assay 24 h post-infection. Antibodies to eEF1A or eIF3A (negative control), and RSV N and P were used in conjunction with Duolink II PLA probes to detect significant proximity between RSV N, P and eEF1A, and no significant proximity between RSV N, P and eIF3A. ( A ) Images were captured on a confocal microscope and ( B ) the number of signals (foci) detected in 100 cells per reaction was quantified using the Duolink Imagetool software (Olink Biosciences). Data was collected by two readers and the mean number of signals/cell was then calculated. Experiments were repeated three times. Mean values, with SEM. Significance identified using 2-way ANOVA. **P

    Techniques Used: Infection, Proximity Assay, Negative Control, Proximity Ligation Assay, Microscopy, Software

    RSV nucleocapsid (N), phosphoprotein (P) and matrix (M) bind to eEF1A in a live virus infection. ( A ) HEK293T or ( B ) A549 cells were infected with RSV at a MOI of 1 pfu/cell and lysed 48 h post-infection. The lysate was incubated with beads bound with antibodies to either eEF1A or eIF3A (negative control). Western blot analysis was performed on lysates before and after immunoprecipitation using antibodies to RSV or eEF1A. A representative blot of immunoprecipitation performed 3 times with consistent results is shown.
    Figure Legend Snippet: RSV nucleocapsid (N), phosphoprotein (P) and matrix (M) bind to eEF1A in a live virus infection. ( A ) HEK293T or ( B ) A549 cells were infected with RSV at a MOI of 1 pfu/cell and lysed 48 h post-infection. The lysate was incubated with beads bound with antibodies to either eEF1A or eIF3A (negative control). Western blot analysis was performed on lysates before and after immunoprecipitation using antibodies to RSV or eEF1A. A representative blot of immunoprecipitation performed 3 times with consistent results is shown.

    Techniques Used: Infection, Incubation, Negative Control, Western Blot, Immunoprecipitation

    17) Product Images from "BAP1 suppresses lung cancer progression and is inhibited by miR-31"

    Article Title: BAP1 suppresses lung cancer progression and is inhibited by miR-31

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7328

    Effects of miR-31 and BAP1 on the growth of lung cancer cell xenografts in mice A. Flow chart of the experimental plan. A549 cells were infected with a control lentivirus or a lentivirus to overexpress miR-31, transfected with a BAP1 overexpression plasmid, or co-transfected with a lentivirus to overexpress miR-31 and a BAP1 overexpression plasmid. A549 cells (2 × 10 6 cells per 0.1 mL) with different treatments were implanted subcutaneously into 6-week-old SCID mice (5 mice per group), and the tumor growth was evaluated on day 35 after cell implantation. B. Representative images of the tumors from the implanted mice. C. Quantitative analysis of the tumor weights. D. Quantitative RT-PCR analysis of miR-31 levels in the tumors from implanted mice. E. Quantitative RT-PCR analysis of BAP1 mRNA levels in the tumors from implanted mice. F. and G. Western blotting analysis of BAP1 protein levels in the tumors from implanted mice. F. representative image; G. quantitative analysis. H. - J. H E-stained sections and immunohistochemical staining for Ki-67 and BAP1 in the tumors from implanted mice. H. representative image; I. and J. quantitative analysis. (* p
    Figure Legend Snippet: Effects of miR-31 and BAP1 on the growth of lung cancer cell xenografts in mice A. Flow chart of the experimental plan. A549 cells were infected with a control lentivirus or a lentivirus to overexpress miR-31, transfected with a BAP1 overexpression plasmid, or co-transfected with a lentivirus to overexpress miR-31 and a BAP1 overexpression plasmid. A549 cells (2 × 10 6 cells per 0.1 mL) with different treatments were implanted subcutaneously into 6-week-old SCID mice (5 mice per group), and the tumor growth was evaluated on day 35 after cell implantation. B. Representative images of the tumors from the implanted mice. C. Quantitative analysis of the tumor weights. D. Quantitative RT-PCR analysis of miR-31 levels in the tumors from implanted mice. E. Quantitative RT-PCR analysis of BAP1 mRNA levels in the tumors from implanted mice. F. and G. Western blotting analysis of BAP1 protein levels in the tumors from implanted mice. F. representative image; G. quantitative analysis. H. - J. H E-stained sections and immunohistochemical staining for Ki-67 and BAP1 in the tumors from implanted mice. H. representative image; I. and J. quantitative analysis. (* p

    Techniques Used: Mouse Assay, Flow Cytometry, Infection, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry

    BAP1 is a direct target of miR-31 A. Quantitative RT-PCR analysis of the expression levels of miR-31 in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic (pre-miR-31), miR-31 inhibitor (anti-miR-31) or scrambled negative control RNA (pre-miR-control or anti-miR-control). B. and C. Western blotting analysis to detect BAP1 protein levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. B: representative image; C. quantitative analysis. D. Quantitative RT-PCR analysis of BAP1 mRNA levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. E. Direct recognition of the BAP1 3′-UTR by miR-31. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-31 binding sites in the BAP1 3′-UTR were co-transfected into A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-31-transfected cells normalized to the negative control RNA-transfected cells. The results are presented as the mean ± S.D. of three independent experiments. (* p
    Figure Legend Snippet: BAP1 is a direct target of miR-31 A. Quantitative RT-PCR analysis of the expression levels of miR-31 in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic (pre-miR-31), miR-31 inhibitor (anti-miR-31) or scrambled negative control RNA (pre-miR-control or anti-miR-control). B. and C. Western blotting analysis to detect BAP1 protein levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. B: representative image; C. quantitative analysis. D. Quantitative RT-PCR analysis of BAP1 mRNA levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. E. Direct recognition of the BAP1 3′-UTR by miR-31. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-31 binding sites in the BAP1 3′-UTR were co-transfected into A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-31-transfected cells normalized to the negative control RNA-transfected cells. The results are presented as the mean ± S.D. of three independent experiments. (* p

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Western Blot, Luciferase, Mutagenesis, Binding Assay, Activity Assay

    Effect of miR-31 and BAP1 on the proliferation and apoptosis of lung cancer cells A. A cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 mimic or scrambled negative control RNA. B. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 inhibitor or scrambled negative control RNA. C. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. and E. The apoptosis assay was performed 24 hours after the transfection of A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA or with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. representative image; E. quantitative analysis. The results of cell proliferation are presented as the mean ± S.E. of three independent experience, and other results are presented as the mean ± S.D. of three independent experiments. (* p
    Figure Legend Snippet: Effect of miR-31 and BAP1 on the proliferation and apoptosis of lung cancer cells A. A cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 mimic or scrambled negative control RNA. B. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 inhibitor or scrambled negative control RNA. C. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. and E. The apoptosis assay was performed 24 hours after the transfection of A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA or with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. representative image; E. quantitative analysis. The results of cell proliferation are presented as the mean ± S.E. of three independent experience, and other results are presented as the mean ± S.D. of three independent experiments. (* p

    Techniques Used: Proliferation Assay, Transfection, Negative Control, Plasmid Preparation, Over Expression, Apoptosis Assay

    18) Product Images from "lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress"

    Article Title: lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

    Journal: Nature cell biology

    doi: 10.1038/ncb3328

    Energy stress induces NBR2 expression through the LKB1-AMPK pathway (a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d) . (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1 . Unprocessed original scans of blots are shown in Supplemental Fig. 8 .
    Figure Legend Snippet: Energy stress induces NBR2 expression through the LKB1-AMPK pathway (a, b) Various cell lines were cultured in 0 or 25 mM glucose-containing medium (a), or 0 or 5 mM 2DG-containing medium (b) for 12–24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c, d) Hela or A549 cells stably expressing EV (empty vector) or Lkb1 expression vectors were cultured in 25 or 0 mM glucose-containing medium, and then subjected to real-time PCR (c) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test) and Western blotting analyses (d) . (e) MDA-MB-231 cells treated with 100 µM A769662 were subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (f) MDA-MB-231 cells were treated with 20 µM Compound C in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 expression (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) MDA-MB-231 cells transfected with AMPKα or control (Ctrl) siRNA were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and then subjected to real-time PCR analysis to measure NBR2 (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for a, b, c, e, f, g can be found in Supplementary Table 1 . Unprocessed original scans of blots are shown in Supplemental Fig. 8 .

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Two Tailed Test, Stable Transfection, Plasmid Preparation, Western Blot, Multiple Displacement Amplification, Transfection

    19) Product Images from "Identification of Preferentially Expressed Antigen of Melanoma as a Potential Tumor Suppressor in Lung Adenocarcinoma"

    Article Title: Identification of Preferentially Expressed Antigen of Melanoma as a Potential Tumor Suppressor in Lung Adenocarcinoma

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.895642

    Cell proliferation of PRAME siRNA-treated cells. MTT assay of PC9 cells ( A ) and A549 cells ( B ) after PRAME (siPRAME) and control siRNA (siN) transfection. * p
    Figure Legend Snippet: Cell proliferation of PRAME siRNA-treated cells. MTT assay of PC9 cells ( A ) and A549 cells ( B ) after PRAME (siPRAME) and control siRNA (siN) transfection. * p

    Techniques Used: MTT Assay, Transfection

    Changes in PRAME expression after siRNA transfection. Messenger RNA ( A ) and protein expression ( B ) are decreased in PC9 cells transfected with PRAME siRNA (siPRAME) compared with that in control siRNA-treated group (siN). Messenger RNA ( C ) and protein expression ( D ) are decreased in A549 cells transfected with PRAME siRNA (siPRAME). Actin serves as the loading control in the experiments. * p
    Figure Legend Snippet: Changes in PRAME expression after siRNA transfection. Messenger RNA ( A ) and protein expression ( B ) are decreased in PC9 cells transfected with PRAME siRNA (siPRAME) compared with that in control siRNA-treated group (siN). Messenger RNA ( C ) and protein expression ( D ) are decreased in A549 cells transfected with PRAME siRNA (siPRAME). Actin serves as the loading control in the experiments. * p

    Techniques Used: Expressing, Transfection

    20) Product Images from "Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1"

    Article Title: Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049744

    SFN suppresses mPGES expression and PGE2 production driven by overexpression of HIF1α. A549 cells were transfected with indicated amounts of HIF-1α and GFP expression vectors. The total amount of transfected DNA (4 µg) was kept constant by addition of the empty vector. The cells were then left untreated or treated with increasing concentrations of SFN for 40 hours. The total cell extracts were prepared for immunoblot analysis to evaluate protein levels. The cell culture media were used for measuring PGE2 concentration by EIA assay.
    Figure Legend Snippet: SFN suppresses mPGES expression and PGE2 production driven by overexpression of HIF1α. A549 cells were transfected with indicated amounts of HIF-1α and GFP expression vectors. The total amount of transfected DNA (4 µg) was kept constant by addition of the empty vector. The cells were then left untreated or treated with increasing concentrations of SFN for 40 hours. The total cell extracts were prepared for immunoblot analysis to evaluate protein levels. The cell culture media were used for measuring PGE2 concentration by EIA assay.

    Techniques Used: Expressing, Over Expression, Transfection, Plasmid Preparation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    SFN reduces mPGES-1 activity in treated cells but does not inhibit activity in purified microsomes. A, SFN treatment of intact cells inhibited mPGES-1 activity, A549 cells were pretreated with or without different concentrations of SFN for 30 minutes and then 1 ng/ml IL1b was added for another 24 hours. mPGES-1 activity was assayed as conversion of PGH2 to PGE2 by microsomal fractions in a mass-spectrometry assay with PGE2(D4) as internal standard. The chromatographic peak height under the m/z = 351.2 ion elution was normalized by internal standard (PGE2d4) peak height under the m/z = 355.2. *, P
    Figure Legend Snippet: SFN reduces mPGES-1 activity in treated cells but does not inhibit activity in purified microsomes. A, SFN treatment of intact cells inhibited mPGES-1 activity, A549 cells were pretreated with or without different concentrations of SFN for 30 minutes and then 1 ng/ml IL1b was added for another 24 hours. mPGES-1 activity was assayed as conversion of PGH2 to PGE2 by microsomal fractions in a mass-spectrometry assay with PGE2(D4) as internal standard. The chromatographic peak height under the m/z = 351.2 ion elution was normalized by internal standard (PGE2d4) peak height under the m/z = 355.2. *, P

    Techniques Used: Activity Assay, Purification, Mass Spectrometry

    SFN dose dependently inhibits IL1β induced PGE2 production. A549 cells were pretreated for 30 minutes in the presence or absence of SFN and then cultured with or without 1 ng/ml IL1β for 24 hours. PGE2 in the media was measured using EIA assay. *, p
    Figure Legend Snippet: SFN dose dependently inhibits IL1β induced PGE2 production. A549 cells were pretreated for 30 minutes in the presence or absence of SFN and then cultured with or without 1 ng/ml IL1β for 24 hours. PGE2 in the media was measured using EIA assay. *, p

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    SFN inhibits mPGES-1 expression and PGE2 production in tumors in vivo . 2.5 × 10 6 A549 cells were injected subcutaneously on the flank of athymic nude mice. After 6 weeks, animals were treated with PBS vehicle (control) or 0.5 mg SFN by intraperitoneal injection and sacrificed 24 hours later. Tumor tissue samples were weighed and frozen for subsequent RT-PCR assay (A), immunoblot analysis (B) and PGE2 measurement (C). The results in A and B are expressed as fold changes relative to PBS treated mice (mean ± SEM; n = 4). *, P
    Figure Legend Snippet: SFN inhibits mPGES-1 expression and PGE2 production in tumors in vivo . 2.5 × 10 6 A549 cells were injected subcutaneously on the flank of athymic nude mice. After 6 weeks, animals were treated with PBS vehicle (control) or 0.5 mg SFN by intraperitoneal injection and sacrificed 24 hours later. Tumor tissue samples were weighed and frozen for subsequent RT-PCR assay (A), immunoblot analysis (B) and PGE2 measurement (C). The results in A and B are expressed as fold changes relative to PBS treated mice (mean ± SEM; n = 4). *, P

    Techniques Used: Expressing, In Vivo, Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    SFN suppresses mPGES-1 expression by inhibiting mRNA transcription. A B. A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot with mPGES-1 antibody. Immunoblot shown (A) is representative of three experiments, that are quantified in panel B, with results expressed as mean fold change ± SEM (n = 3). C D, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added (C) or not (D) and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as mean fold change ± SEM (n = 3). *, P
    Figure Legend Snippet: SFN suppresses mPGES-1 expression by inhibiting mRNA transcription. A B. A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot with mPGES-1 antibody. Immunoblot shown (A) is representative of three experiments, that are quantified in panel B, with results expressed as mean fold change ± SEM (n = 3). C D, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added (C) or not (D) and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as mean fold change ± SEM (n = 3). *, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR

    SFN dose dependently inhibits HIF-1α and Pol II occupancy at the mPGES-1 promoter. A549 cells were pretreated with or without SFN and HIF-1α inhibitor 2-Methoxyestradiol (2ME2, 0.2 mM) for 30 minutes. IL1b (1 ng/ml) was either added (left panels) or not (right panels) and cells were cultured for another 4 hours. ChIP assays, coupled with quantitative real time PCR, were performed using HIF-1α (A), Pol II (B) or Egr-1(C) antibodies. The results are expressed as fold change of protein binding to the mPGES-1 promoter relative to untreated cells. (mean ± SEM; n = 3) *, P
    Figure Legend Snippet: SFN dose dependently inhibits HIF-1α and Pol II occupancy at the mPGES-1 promoter. A549 cells were pretreated with or without SFN and HIF-1α inhibitor 2-Methoxyestradiol (2ME2, 0.2 mM) for 30 minutes. IL1b (1 ng/ml) was either added (left panels) or not (right panels) and cells were cultured for another 4 hours. ChIP assays, coupled with quantitative real time PCR, were performed using HIF-1α (A), Pol II (B) or Egr-1(C) antibodies. The results are expressed as fold change of protein binding to the mPGES-1 promoter relative to untreated cells. (mean ± SEM; n = 3) *, P

    Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Protein Binding

    SFN does not inhibit COX-2 expression in A549 cells. A, A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added (left) or not (right) and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot analysis. B, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). C, A549 cells were treated with 1 ng/ml IL1β either with or without 10 µM SFN for different times. Total cell lysates were subjected to immunoblot analysis.
    Figure Legend Snippet: SFN does not inhibit COX-2 expression in A549 cells. A, A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added (left) or not (right) and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot analysis. B, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). C, A549 cells were treated with 1 ng/ml IL1β either with or without 10 µM SFN for different times. Total cell lysates were subjected to immunoblot analysis.

    Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    SFN inhibits HIF-1α protein accumulation. Cell lines were treated as described below, and total cell lysates subjected to immunoblot analysis. A, A549 cells were pretreated with or without SFN and MG132 (20 µM) for 1 hour. B, MCF7 cells were pretreated for 1 hour with indicated concentration of SFN, and then treated with 100µM cobalt chloride (Co +2 ) for an additional 5.5hours. C, 4T1 cells were pretreated for 20 min with 20 µM SFN, followed by treatment with 250µM cobalt chloride or 200µM desferroxamine as indicated for 4hours. D. 293 cells were pretreated for 20 min with indicated concentrations of SFN, followed by stimulation with 250 µM cobalt chloride for an additional 4 hours.
    Figure Legend Snippet: SFN inhibits HIF-1α protein accumulation. Cell lines were treated as described below, and total cell lysates subjected to immunoblot analysis. A, A549 cells were pretreated with or without SFN and MG132 (20 µM) for 1 hour. B, MCF7 cells were pretreated for 1 hour with indicated concentration of SFN, and then treated with 100µM cobalt chloride (Co +2 ) for an additional 5.5hours. C, 4T1 cells were pretreated for 20 min with 20 µM SFN, followed by treatment with 250µM cobalt chloride or 200µM desferroxamine as indicated for 4hours. D. 293 cells were pretreated for 20 min with indicated concentrations of SFN, followed by stimulation with 250 µM cobalt chloride for an additional 4 hours.

    Techniques Used: Concentration Assay

    SFN suppresses the HIF-1–mPGES –PGE2 axis by control of HIF-1α protein expression without altering mRNA levels. A, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). B C, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added or not and cells were cultured for another 4 hours. Total cell lysates were subjected to immunoblot analysis (panel B) and total RNA was analyzed by quantitative RT-PCR (panel C).
    Figure Legend Snippet: SFN suppresses the HIF-1–mPGES –PGE2 axis by control of HIF-1α protein expression without altering mRNA levels. A, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). B C, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added or not and cells were cultured for another 4 hours. Total cell lysates were subjected to immunoblot analysis (panel B) and total RNA was analyzed by quantitative RT-PCR (panel C).

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR

    21) Product Images from "Nicotine-induced survival signaling in lung cancer cells is dependent on their p53 status while its down-regulation by curcumin is independent"

    Article Title: Nicotine-induced survival signaling in lung cancer cells is dependent on their p53 status while its down-regulation by curcumin is independent

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-220

    Nicotine induces proliferation and NF-κB nuclear translocation in Hop-92, NCI-H522 and p53-silenced A549 cells . (A-B) The cells were treated with nicotine and/or curcumin and cell viability was measured by MTT assay. All error bars indicate standard deviation between three independent experiments. (C-D) Nuclear extracts were prepared from the cells treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. (E) A549 cells were transfected with indicated concentrations of control-si RNA or p53-si RNA and after 48 h, the whole cell lysates were Western blotted against p53 antibody. (F) A549 cells were transfected with indicated concentrations of control-si RNA or p53-si RNA and after 48 h, treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. All blots and EMSAs are representative samples of three independent experiments.
    Figure Legend Snippet: Nicotine induces proliferation and NF-κB nuclear translocation in Hop-92, NCI-H522 and p53-silenced A549 cells . (A-B) The cells were treated with nicotine and/or curcumin and cell viability was measured by MTT assay. All error bars indicate standard deviation between three independent experiments. (C-D) Nuclear extracts were prepared from the cells treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. (E) A549 cells were transfected with indicated concentrations of control-si RNA or p53-si RNA and after 48 h, the whole cell lysates were Western blotted against p53 antibody. (F) A549 cells were transfected with indicated concentrations of control-si RNA or p53-si RNA and after 48 h, treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. All blots and EMSAs are representative samples of three independent experiments.

    Techniques Used: Translocation Assay, MTT Assay, Standard Deviation, Transfection, Western Blot

    Curcumin inhibits enhancement of clonogenicity by nicotine, which induces p53 and p21 in A549 and p21 in H1299-p53 WT . (A-B) The cells were treated with nicotine and/or curcumin for 72 h, after which cells were split and seeded in six well plates, kept for 2 weeks and clones developed were stained and counted. (C-E) The cells were treated with nicotine (10 -9 -10 -2 M) for 24 h and whole cell lysates were Western blotted against p53 antibody. All figures are representative samples of three independent experiments.
    Figure Legend Snippet: Curcumin inhibits enhancement of clonogenicity by nicotine, which induces p53 and p21 in A549 and p21 in H1299-p53 WT . (A-B) The cells were treated with nicotine and/or curcumin for 72 h, after which cells were split and seeded in six well plates, kept for 2 weeks and clones developed were stained and counted. (C-E) The cells were treated with nicotine (10 -9 -10 -2 M) for 24 h and whole cell lysates were Western blotted against p53 antibody. All figures are representative samples of three independent experiments.

    Techniques Used: Clone Assay, Staining, Western Blot

    Nicotine activates NF-κB in H1299, but not in A549 . (A-B) Nuclear extracts were prepared from the cells treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. (C) H1299 cells were treated with 10 -8 M nicotine for different time intervals and EMSA was done. (D) The cytosolic extracts of the time kinetic study of NF-κB activation were Western blotted against the respective antibodies. (E) The nuclear extract of nicotine-treated H1299 cells were incubated with p50 and p65 antibodies or unlabelled oligo and EMSA was conducted to detect supershift and cold competition respectively. (F) H1299 cells were treated with curcumin and then with nicotine for 30 min and EMSA was done. All blots and EMSAs are representative samples of three independent experiments.
    Figure Legend Snippet: Nicotine activates NF-κB in H1299, but not in A549 . (A-B) Nuclear extracts were prepared from the cells treated with indicated concentrations of nicotine for 30 min and NF-κB nuclear translocation was assessed by EMSA. (C) H1299 cells were treated with 10 -8 M nicotine for different time intervals and EMSA was done. (D) The cytosolic extracts of the time kinetic study of NF-κB activation were Western blotted against the respective antibodies. (E) The nuclear extract of nicotine-treated H1299 cells were incubated with p50 and p65 antibodies or unlabelled oligo and EMSA was conducted to detect supershift and cold competition respectively. (F) H1299 cells were treated with curcumin and then with nicotine for 30 min and EMSA was done. All blots and EMSAs are representative samples of three independent experiments.

    Techniques Used: Translocation Assay, Activation Assay, Western Blot, Incubation

    Over-expression and silencing of p53 produce reciprocal effect on the activation pattern of NF-κB, MAPKs and AP-1 . (A-B) H1299 and A549 cells were transfected with pcDNA3-p53 WT and pcDNA3-p53 DN constructs respectively and the stable clones selected were lysed and Western blotted against p53 antibody. (C-D) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done using nuclear extracts. (E) A549 cells were treated with different concentrations of pifithrin-α (10-50 μM) for 4 h and the whole cell lyasates prepared were Western blotted against p53 antibody. (F) A549 cells were treated with 40 μM pifithrin-α for 4 h followed by nicotine (10 -9 -10 -2 M) for 30 min and EMSA was done. (G-H) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done. (I-J) H1299 WT-p53 and A549 DN-p53 cells were treated with nicotine (10 -9 -10 -2 M) for 30 min and whole cell lysates were Western blotted against respective phospho specific antibodies. All blots and EMSAs are representative samples of three independent experiments.
    Figure Legend Snippet: Over-expression and silencing of p53 produce reciprocal effect on the activation pattern of NF-κB, MAPKs and AP-1 . (A-B) H1299 and A549 cells were transfected with pcDNA3-p53 WT and pcDNA3-p53 DN constructs respectively and the stable clones selected were lysed and Western blotted against p53 antibody. (C-D) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done using nuclear extracts. (E) A549 cells were treated with different concentrations of pifithrin-α (10-50 μM) for 4 h and the whole cell lyasates prepared were Western blotted against p53 antibody. (F) A549 cells were treated with 40 μM pifithrin-α for 4 h followed by nicotine (10 -9 -10 -2 M) for 30 min and EMSA was done. (G-H) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done. (I-J) H1299 WT-p53 and A549 DN-p53 cells were treated with nicotine (10 -9 -10 -2 M) for 30 min and whole cell lysates were Western blotted against respective phospho specific antibodies. All blots and EMSAs are representative samples of three independent experiments.

    Techniques Used: Over Expression, Activation Assay, Transfection, Construct, Clone Assay, Western Blot

    Nicotine induces more proliferation in H1299 than in A549, which is down regulated by curcumin . (A-B) The cells were treated with nicotine and/or curcumin for 72 h and 24 h respectively for MTT assay and thymidine incorporation assay. Tritiated thymidine (0.5 μCi/well) was added 6 h before the completion of incubation. All error bars indicate standard deviation between three independent experiments.
    Figure Legend Snippet: Nicotine induces more proliferation in H1299 than in A549, which is down regulated by curcumin . (A-B) The cells were treated with nicotine and/or curcumin for 72 h and 24 h respectively for MTT assay and thymidine incorporation assay. Tritiated thymidine (0.5 μCi/well) was added 6 h before the completion of incubation. All error bars indicate standard deviation between three independent experiments.

    Techniques Used: MTT Assay, Thymidine Incorporation Assay, Incubation, Standard Deviation

    22) Product Images from "Five birds, one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody"

    Article Title: Five birds, one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

    Journal: mAbs

    doi: 10.4161/19420862.2014.985132

    In vitro potency of recombinant S. aureus toxins. Cytolytic activity of indicated toxins was measured using ( A ): A549 cells, ( B ): human PMNs, ( C ): human RBCs or ( D ): rabbit RBCs in the indicated concentration range. Error bars indicate mean +/− SEM, n = 2.
    Figure Legend Snippet: In vitro potency of recombinant S. aureus toxins. Cytolytic activity of indicated toxins was measured using ( A ): A549 cells, ( B ): human PMNs, ( C ): human RBCs or ( D ): rabbit RBCs in the indicated concentration range. Error bars indicate mean +/− SEM, n = 2.

    Techniques Used: In Vitro, Recombinant, Activity Assay, Concentration Assay

    23) Product Images from "MicroRNA-374b-5p Functions as a Tumor Suppressor in Non-Small Cell Lung Cancer by Targeting FOXP1 and Predicts Prognosis of Cancer Patients"

    Article Title: MicroRNA-374b-5p Functions as a Tumor Suppressor in Non-Small Cell Lung Cancer by Targeting FOXP1 and Predicts Prognosis of Cancer Patients

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S243221

    Effects of miR-374b-5p on cell proliferation, migration and invasion of A549 and H460 cells. ( A ) Cell transfection efficiency examination results. ( B ) Inhibiting effect of miR-374b-5p on NSCLC cell proliferation. ( C and D) miR-374b-5p suppressed NSCLC cell migration ( C ) and invasion ( D ). * P
    Figure Legend Snippet: Effects of miR-374b-5p on cell proliferation, migration and invasion of A549 and H460 cells. ( A ) Cell transfection efficiency examination results. ( B ) Inhibiting effect of miR-374b-5p on NSCLC cell proliferation. ( C and D) miR-374b-5p suppressed NSCLC cell migration ( C ) and invasion ( D ). * P

    Techniques Used: Migration, Transfection

    24) Product Images from "Glucocorticoid Receptor-mediated transactivation is hampered by Striatin-3, a novel interaction partner of the receptor"

    Article Title: Glucocorticoid Receptor-mediated transactivation is hampered by Striatin-3, a novel interaction partner of the receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09246-6

    DEX treatment of A549 cells supports co-detection of endogenous GR and STRN3 in the nucleus. A549 cells were seeded on coverslips and incubated in phenol-red free and serum-free medium for 4 h. Cells were treated with either solvent or 1 µM of DEX, for 2 h following by indirect immunofluorescence analysis to detect GR (green signal) and/or STRN3 (red signal). Cell nuclei are visualized using DAPI (blue signal). Assessment of co-localization of the protein signal for GR and STRN3 (middle frame) was performed by correlation statistics using the Olympus Fluoview version 4.2 software. The image is a representative result of 5 recorded images, with each field containing minimally 10 cells.
    Figure Legend Snippet: DEX treatment of A549 cells supports co-detection of endogenous GR and STRN3 in the nucleus. A549 cells were seeded on coverslips and incubated in phenol-red free and serum-free medium for 4 h. Cells were treated with either solvent or 1 µM of DEX, for 2 h following by indirect immunofluorescence analysis to detect GR (green signal) and/or STRN3 (red signal). Cell nuclei are visualized using DAPI (blue signal). Assessment of co-localization of the protein signal for GR and STRN3 (middle frame) was performed by correlation statistics using the Olympus Fluoview version 4.2 software. The image is a representative result of 5 recorded images, with each field containing minimally 10 cells.

    Techniques Used: Incubation, Immunofluorescence, Software

    PPP2CA mediates the effect of STRN3 on GR phosphorylation. ( A ) Identification of common interacting phosphatases for STRN3 and GR using Ingenuity Pathway Analysis (IPA) database. ( B ) A549 cells were transfected in 10 cm plates with 50 nM of control (lanes 1–4) or with PPP2CA (lanes 5–8) siRNA. After 24 h, Flag-STRN3 (lanes 3–4, 7–8) or mock (lanes 1–2, 5–6) plasmids were over-expressed (6 μ of total DNA was transfected per plate). After 48 h, the cells were treated with vehicle or 1 μM Dex for 2 h and lysed. The nuclear fractions of the lysates were analyzed by immunoblotting against GR phosphorylated at S211, total GR (86 kDa), (Flag-) STRN3 (96 kDa), PPP2CA (35 kDa) and PARP (116 kDa, nuclear marker). Total GR expression was identified in a separate immunoblot to avoid multiple stripping of the membrane. This is a representative experiment out of three independent experiments that have been performed with similar results. ( C ) Co-immunoprecipitation experiment in HEK293T cells for the complexes GR/STRN3/PPP2CA, GR/PPP2CA and STRN3/PPP2CA (in 10 cm plates). Over-expression of 4 μg PPP2CA (lane 1) and over-expression of 4 μg CFP-GR (lane 2) serve as negative controls. Additionally, the cells were transfected with 3 μg Flag-STRN3, 4 μg CFP-GR and 4 μg PPP2CA (lanes 3–4) or with 3 μg Flag-GR and 4 μg PPP2CA (lanes 5–6) or with 3 μg Flag-STRN3 and 4 μg PPP2CA (lanes 7–8). 48 h after transfection, the cells were treated with vehicle or with 1 μM Dex as indicated (2 h). The lysates were used for IP assays using M2-Flag beads and the precipitates were analyzed with immunoblotting against (Flag-) STRN3 (96 kDa), (CFP- or Flag-) GR (117 or 86 kDa, respectively) and PPP2CA (35 kDa) (11 μg of total DNA was transfected per plate) (full-length blots of ( B,C ) are provided in Supplementary S16 and S17 , respectively). ( D ) The complexes Flag-GR/YFP-STRN3/PPP2CA and Flag-GR/PPP2CA were examined for their phosphatase activity. HEK293T cells (in 10 cm plates) were transfected with 4 μg PPP2CA (negative control) or with 3 μg Flag-GR, 4 μg YFP-STRN3 and 4 μg PPP2CA or with 3 μg Flag-GR and 4 μg PPP2CA. All the samples were treated with 1 μM Dex for 2 h. The complexes were precipitated with M2-Flag beads and then incubated with Flag peptide in order to detach the complexes from the beads. Then the samples were processed for the phosphatase assay (the assay is described in the section of Methods). Additional negative controls: no-substrate condition (diluent) and total lysate from cells stimulated with 100 nM Okadaic acid, a phosphatase inhibitor. Positive control: total cell lysate (11 μg of total DNA was transfected per plate). B, C and D are representations of a series of triplicate experiments that all yielded similar results.
    Figure Legend Snippet: PPP2CA mediates the effect of STRN3 on GR phosphorylation. ( A ) Identification of common interacting phosphatases for STRN3 and GR using Ingenuity Pathway Analysis (IPA) database. ( B ) A549 cells were transfected in 10 cm plates with 50 nM of control (lanes 1–4) or with PPP2CA (lanes 5–8) siRNA. After 24 h, Flag-STRN3 (lanes 3–4, 7–8) or mock (lanes 1–2, 5–6) plasmids were over-expressed (6 μ of total DNA was transfected per plate). After 48 h, the cells were treated with vehicle or 1 μM Dex for 2 h and lysed. The nuclear fractions of the lysates were analyzed by immunoblotting against GR phosphorylated at S211, total GR (86 kDa), (Flag-) STRN3 (96 kDa), PPP2CA (35 kDa) and PARP (116 kDa, nuclear marker). Total GR expression was identified in a separate immunoblot to avoid multiple stripping of the membrane. This is a representative experiment out of three independent experiments that have been performed with similar results. ( C ) Co-immunoprecipitation experiment in HEK293T cells for the complexes GR/STRN3/PPP2CA, GR/PPP2CA and STRN3/PPP2CA (in 10 cm plates). Over-expression of 4 μg PPP2CA (lane 1) and over-expression of 4 μg CFP-GR (lane 2) serve as negative controls. Additionally, the cells were transfected with 3 μg Flag-STRN3, 4 μg CFP-GR and 4 μg PPP2CA (lanes 3–4) or with 3 μg Flag-GR and 4 μg PPP2CA (lanes 5–6) or with 3 μg Flag-STRN3 and 4 μg PPP2CA (lanes 7–8). 48 h after transfection, the cells were treated with vehicle or with 1 μM Dex as indicated (2 h). The lysates were used for IP assays using M2-Flag beads and the precipitates were analyzed with immunoblotting against (Flag-) STRN3 (96 kDa), (CFP- or Flag-) GR (117 or 86 kDa, respectively) and PPP2CA (35 kDa) (11 μg of total DNA was transfected per plate) (full-length blots of ( B,C ) are provided in Supplementary S16 and S17 , respectively). ( D ) The complexes Flag-GR/YFP-STRN3/PPP2CA and Flag-GR/PPP2CA were examined for their phosphatase activity. HEK293T cells (in 10 cm plates) were transfected with 4 μg PPP2CA (negative control) or with 3 μg Flag-GR, 4 μg YFP-STRN3 and 4 μg PPP2CA or with 3 μg Flag-GR and 4 μg PPP2CA. All the samples were treated with 1 μM Dex for 2 h. The complexes were precipitated with M2-Flag beads and then incubated with Flag peptide in order to detach the complexes from the beads. Then the samples were processed for the phosphatase assay (the assay is described in the section of Methods). Additional negative controls: no-substrate condition (diluent) and total lysate from cells stimulated with 100 nM Okadaic acid, a phosphatase inhibitor. Positive control: total cell lysate (11 μg of total DNA was transfected per plate). B, C and D are representations of a series of triplicate experiments that all yielded similar results.

    Techniques Used: Indirect Immunoperoxidase Assay, Transfection, Marker, Expressing, Stripping Membranes, Immunoprecipitation, Over Expression, Activity Assay, Negative Control, Incubation, Phosphatase Assay, Positive Control

    STRN3 is an inhibitor of GR trans-activation. ( A ) HEKT293T cells were transfected in 96-well plate with 30 ng of Flag-GR, 15 ng of GRE-luc and over-expression of STRN3, 30 ng and 60 ng, as indicated (or empty vector so that total transfected DNA is 110 ng). Cells were treated with 1 μM Dex or vehicle, for 6 h. In the last two lanes, Flag-GR was not over-expressed. ( B ) A549 cells stably transfected with GRE-Luc reporter were transfected with 50 nM siRNA On Target plus (Dharmacon) for human STRN3. Cells were treated with 1 μM Dex or vehicle for 24 h and processed for luciferase measurement. ( C ) Representation of the absolute luciferase values of graph in ( B ). ( D ) WT A549 cells were transfected with STRN3-specific siRNA or control siRNA (as in B and C ) and stimulated with 1 μM Dex or vehicle for 6 h before lysis and RNA extraction. A549 cells treated as in ( B , C ) were processed for qPCR to determine the endogenous STRN3 levels and ( E ) the levels of the GR-dependent genes DUSP1, PER1, ZFP and ECI2. ( F ) A549 cells stably transfected with NF-κB-Luc reporter are transfected with human STRN3-specific siRNA or control siRNA (as in B , C , E ). Cells are subsequently treated with vehicle or with 1000 U/ml of human TNF or pre-treated with 1000 U/ml of human TNF for 1 h followed by 1 μM Dex for 6 h or with 1 μM Dex alone for 6 h. All data are represented as fold change to the vehicle-treated condition. The graphs represent pooled data from three independent experiments.
    Figure Legend Snippet: STRN3 is an inhibitor of GR trans-activation. ( A ) HEKT293T cells were transfected in 96-well plate with 30 ng of Flag-GR, 15 ng of GRE-luc and over-expression of STRN3, 30 ng and 60 ng, as indicated (or empty vector so that total transfected DNA is 110 ng). Cells were treated with 1 μM Dex or vehicle, for 6 h. In the last two lanes, Flag-GR was not over-expressed. ( B ) A549 cells stably transfected with GRE-Luc reporter were transfected with 50 nM siRNA On Target plus (Dharmacon) for human STRN3. Cells were treated with 1 μM Dex or vehicle for 24 h and processed for luciferase measurement. ( C ) Representation of the absolute luciferase values of graph in ( B ). ( D ) WT A549 cells were transfected with STRN3-specific siRNA or control siRNA (as in B and C ) and stimulated with 1 μM Dex or vehicle for 6 h before lysis and RNA extraction. A549 cells treated as in ( B , C ) were processed for qPCR to determine the endogenous STRN3 levels and ( E ) the levels of the GR-dependent genes DUSP1, PER1, ZFP and ECI2. ( F ) A549 cells stably transfected with NF-κB-Luc reporter are transfected with human STRN3-specific siRNA or control siRNA (as in B , C , E ). Cells are subsequently treated with vehicle or with 1000 U/ml of human TNF or pre-treated with 1000 U/ml of human TNF for 1 h followed by 1 μM Dex for 6 h or with 1 μM Dex alone for 6 h. All data are represented as fold change to the vehicle-treated condition. The graphs represent pooled data from three independent experiments.

    Techniques Used: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Stable Transfection, Luciferase, Lysis, RNA Extraction, Real-time Polymerase Chain Reaction

    STRN3 mediates the dephosphorylation of GR at serine 211. ( A ) Protein structure of human GR with its respective domains (NTD, DBD, HR, LBD) and the phosphorylation sites indicated (S, serine, T, threonine). ( B ) A549 cells were transfected in 6-wellplate with gradient amounts of YFP-STRN3 plasmid, 400 ng, 800 ng, 1600 ng or mock DNA (empty vector was used so that total transfected DNA is 2 μg/well) and stimulated with vehicle (−) or 1 μM Dex (+) for 2 h. The cells were lysed and processed for immunoblotting against GR phosphorylated at S211 (GR S211), total GR (86 kDa) and GAPDH (36 kDa). ( C ) A549 cells were transfected with 50 nM of STRN3-specific siRNA in 6-wellplate and stimulated with vehicle (−) or with 1 μM Dex (+) for 2 h. Samples were processed either for immunoblotting against GR phosphorylated at S211, total GR and GAPDH (The blots show one representative experiment out of three performed) or for ( D ) RNA isolation and qPCR to determine the endogenous levels of STRN3 and the efficiency of STRN3 silencing (statistics are not performed as data represent a technical triplicate). ( E ) YFP-STRN3 (or mock plasmid, empty vector) was over-expressed in A549 cells at a concentration of 6 μg in 10 cm plates and stimulated with vehicle (−) or with 1 μM Dex (+) for 2 h. Cells were lysed and separated into cytoplasmic and nuclear fractions. Cellular fractions were analyzed by gel electrophoresis and immnunoblotted using antibodies against GR phosphorylated at S211, total GR (86 kDa), (YFP-) STRN3 (122 kDa), Lamin A/C (70 kDa, nuclear marker) and GAPDH (36 kDa, cytoplasmic marker). The blots show one representative experiment out of three performed (full-length blots of 5B, 5C and 5E are provided in Supplementary S12 , S13 and S14 , respectively).
    Figure Legend Snippet: STRN3 mediates the dephosphorylation of GR at serine 211. ( A ) Protein structure of human GR with its respective domains (NTD, DBD, HR, LBD) and the phosphorylation sites indicated (S, serine, T, threonine). ( B ) A549 cells were transfected in 6-wellplate with gradient amounts of YFP-STRN3 plasmid, 400 ng, 800 ng, 1600 ng or mock DNA (empty vector was used so that total transfected DNA is 2 μg/well) and stimulated with vehicle (−) or 1 μM Dex (+) for 2 h. The cells were lysed and processed for immunoblotting against GR phosphorylated at S211 (GR S211), total GR (86 kDa) and GAPDH (36 kDa). ( C ) A549 cells were transfected with 50 nM of STRN3-specific siRNA in 6-wellplate and stimulated with vehicle (−) or with 1 μM Dex (+) for 2 h. Samples were processed either for immunoblotting against GR phosphorylated at S211, total GR and GAPDH (The blots show one representative experiment out of three performed) or for ( D ) RNA isolation and qPCR to determine the endogenous levels of STRN3 and the efficiency of STRN3 silencing (statistics are not performed as data represent a technical triplicate). ( E ) YFP-STRN3 (or mock plasmid, empty vector) was over-expressed in A549 cells at a concentration of 6 μg in 10 cm plates and stimulated with vehicle (−) or with 1 μM Dex (+) for 2 h. Cells were lysed and separated into cytoplasmic and nuclear fractions. Cellular fractions were analyzed by gel electrophoresis and immnunoblotted using antibodies against GR phosphorylated at S211, total GR (86 kDa), (YFP-) STRN3 (122 kDa), Lamin A/C (70 kDa, nuclear marker) and GAPDH (36 kDa, cytoplasmic marker). The blots show one representative experiment out of three performed (full-length blots of 5B, 5C and 5E are provided in Supplementary S12 , S13 and S14 , respectively).

    Techniques Used: De-Phosphorylation Assay, Transfection, Plasmid Preparation, Isolation, Real-time Polymerase Chain Reaction, Concentration Assay, Nucleic Acid Electrophoresis, Marker

    25) Product Images from "Degradable Hollow Mesoporous Silicon/Carbon Nanoparticles for Photoacoustic Imaging-Guided Highly Effective Chemo-Thermal Tumor Therapy in Vitro and in Vivo"

    Article Title: Degradable Hollow Mesoporous Silicon/Carbon Nanoparticles for Photoacoustic Imaging-Guided Highly Effective Chemo-Thermal Tumor Therapy in Vitro and in Vivo

    Journal: Theranostics

    doi: 10.7150/thno.18460

    a) In vivo PA imaging of tumor tissue before and after tail intravenous injection of 250 μL PEG-Si/C-DOX NPs (2 mg/mL) under 800 nm laser irradiation at different time points (0.5, 1, 2, 4, 8 and 12 h), inset is the normalized PA signals in tumor at different time points; b) Infrared thermal images of A549 tumor-bearing mice injected with PBS, the PEG-Si/C NPs and the PEG-Si/C-DOX NPs exposed to 808 nm laser at a power densities of 1 W/cm 2 recorded at different time intervals, respectively; c) Temperature of tumors monitored by the infrared thermal camera in different groups upon laser irradiation as indicated in b).
    Figure Legend Snippet: a) In vivo PA imaging of tumor tissue before and after tail intravenous injection of 250 μL PEG-Si/C-DOX NPs (2 mg/mL) under 800 nm laser irradiation at different time points (0.5, 1, 2, 4, 8 and 12 h), inset is the normalized PA signals in tumor at different time points; b) Infrared thermal images of A549 tumor-bearing mice injected with PBS, the PEG-Si/C NPs and the PEG-Si/C-DOX NPs exposed to 808 nm laser at a power densities of 1 W/cm 2 recorded at different time intervals, respectively; c) Temperature of tumors monitored by the infrared thermal camera in different groups upon laser irradiation as indicated in b).

    Techniques Used: In Vivo, Imaging, Injection, Irradiation, Mouse Assay

    Cellular imaging and in vitro cytotoxicity. a) In vitro cellular imaging and localization of free DOX and different NPs, monitored in A549 cell; b) In vitro combination therapeutic effect of free DOX, the PEG-Si/C NPs, the PEG-Si/C-DOX NPs with or without 808 nm laser irradiation at a power density of 0.5 W/cm 2 for 5 min; c) In vitro cytotoxicity of the PMHC 18 -PEG.
    Figure Legend Snippet: Cellular imaging and in vitro cytotoxicity. a) In vitro cellular imaging and localization of free DOX and different NPs, monitored in A549 cell; b) In vitro combination therapeutic effect of free DOX, the PEG-Si/C NPs, the PEG-Si/C-DOX NPs with or without 808 nm laser irradiation at a power density of 0.5 W/cm 2 for 5 min; c) In vitro cytotoxicity of the PMHC 18 -PEG.

    Techniques Used: Imaging, In Vitro, Irradiation

    In vivo therapeutic efficacy of the PEG-Si/C-DOX NPs. b) Representative photographs of mice bearing A549 tumor after different treatments for varied time periods; c) Relative tumor volumes and d) body weight of mice treated with PBS, PBS + Laser, free DOX, PEG-Si/C NPs + Laser, PEG-Si/C-DOX NPs + Laser; e) Biodistribution of PEG-Si/C-DOX NPs via ICP-AES analysis of the silicon element at different times post-injection.
    Figure Legend Snippet: In vivo therapeutic efficacy of the PEG-Si/C-DOX NPs. b) Representative photographs of mice bearing A549 tumor after different treatments for varied time periods; c) Relative tumor volumes and d) body weight of mice treated with PBS, PBS + Laser, free DOX, PEG-Si/C NPs + Laser, PEG-Si/C-DOX NPs + Laser; e) Biodistribution of PEG-Si/C-DOX NPs via ICP-AES analysis of the silicon element at different times post-injection.

    Techniques Used: In Vivo, Mouse Assay, Injection

    26) Product Images from "CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to–mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding"

    Article Title: CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to–mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18598

    CBP501 inhibits KRas/CaM interaction (A) Cellular lysates (1 mL) from H1299 cells were incubated with CaM-sepharose together with CBP501 (2 μM) or EGTA (1 mM) in the presence of Ca 2+ for 2 h as indicated in Materials and Methods. The presence of KRas and β-actin were analyzed by western blot assay using specific antibodies. (B) Cellular lysates (1 mL) from H1299 cells were incubated with CaM-sepharose (CaM-seph) or with Plain (Seph) beads together with CBP501 (2 μM) or OBA (2 μM) in the presence of Ca 2+ as indicated in Materials and Methods. Cellular lysates (1 mL) from H1299 cells (C) and A549 cells (D) were incubated with CaM-sepharose (CaM-seph) or with Plain (Seph) beads together with CBP501 (2 μM), W7 (20 μM) or CMZ (5 μM) in the presence of Ca 2+ as indicated in Materials and Methods. Cell lysates were analyzed by western blot assay with antibodies to KRas, IQGAP1, p68 and β-actin. The results are the average relative intensity of three replicate blots.
    Figure Legend Snippet: CBP501 inhibits KRas/CaM interaction (A) Cellular lysates (1 mL) from H1299 cells were incubated with CaM-sepharose together with CBP501 (2 μM) or EGTA (1 mM) in the presence of Ca 2+ for 2 h as indicated in Materials and Methods. The presence of KRas and β-actin were analyzed by western blot assay using specific antibodies. (B) Cellular lysates (1 mL) from H1299 cells were incubated with CaM-sepharose (CaM-seph) or with Plain (Seph) beads together with CBP501 (2 μM) or OBA (2 μM) in the presence of Ca 2+ as indicated in Materials and Methods. Cellular lysates (1 mL) from H1299 cells (C) and A549 cells (D) were incubated with CaM-sepharose (CaM-seph) or with Plain (Seph) beads together with CBP501 (2 μM), W7 (20 μM) or CMZ (5 μM) in the presence of Ca 2+ as indicated in Materials and Methods. Cell lysates were analyzed by western blot assay with antibodies to KRas, IQGAP1, p68 and β-actin. The results are the average relative intensity of three replicate blots.

    Techniques Used: Chick Chorioallantoic Membrane Assay, Incubation, Western Blot

    CBP501 attenuates EGF-dependent EMT via PI3K/Akt pathway (A) A549 cells were treated with or without EGF (50 or 100 ng/mL) in combination with CBP501 (1 or 5 μM) for 72 h. Cell lysates were analyzed by western blot assay with antibodies to E-cadherin, Vimentin, Zeb1, and β-actin. The measured band density was normalized relative to that of the control sample, with the control value set to 1. β-actin was used as a loading control. The results are the average relative intensity of three replicate blots. FL , full length; CF , cleaved fragment. (B) A549 cells were serum-starved for 1 day and then pretreated with CBP501 (1 μM) for 3 h before stimulating with EGF. The cells were treated with EGF (100 ng/ml) for 0-60 min. Cell lysates were analyzed by western blot assay ( n = 3) with antibodies to pAkt, Akt, pERK1/2, ERK1/2, pSTAT3, STAT3 and β-actin. (C) Quantification of H1299 cell invasion by EGF stimulation in spheroid invasion assay. Cells were aggregated into spheroids and then induced to invade the surrounding matrix for 7 days with or without EGF (500 ng/mL) stimulation. The total area of each invading spheroid was calculated with Image-J software and taken to be a measure of cell invasion ( n = 3). Red signal threshold was set to capture the total structure. Scale bar is 500 μm. Data, the mean ± SD; * and **, P
    Figure Legend Snippet: CBP501 attenuates EGF-dependent EMT via PI3K/Akt pathway (A) A549 cells were treated with or without EGF (50 or 100 ng/mL) in combination with CBP501 (1 or 5 μM) for 72 h. Cell lysates were analyzed by western blot assay with antibodies to E-cadherin, Vimentin, Zeb1, and β-actin. The measured band density was normalized relative to that of the control sample, with the control value set to 1. β-actin was used as a loading control. The results are the average relative intensity of three replicate blots. FL , full length; CF , cleaved fragment. (B) A549 cells were serum-starved for 1 day and then pretreated with CBP501 (1 μM) for 3 h before stimulating with EGF. The cells were treated with EGF (100 ng/ml) for 0-60 min. Cell lysates were analyzed by western blot assay ( n = 3) with antibodies to pAkt, Akt, pERK1/2, ERK1/2, pSTAT3, STAT3 and β-actin. (C) Quantification of H1299 cell invasion by EGF stimulation in spheroid invasion assay. Cells were aggregated into spheroids and then induced to invade the surrounding matrix for 7 days with or without EGF (500 ng/mL) stimulation. The total area of each invading spheroid was calculated with Image-J software and taken to be a measure of cell invasion ( n = 3). Red signal threshold was set to capture the total structure. Scale bar is 500 μm. Data, the mean ± SD; * and **, P

    Techniques Used: Western Blot, Invasion Assay, Software

    KRas knockdown cells attenuate inhibition of cell migration by CBP501 (A) Stable silencing of KRas in A549 and H1299 cells was assessed by comparing KRas expression in cell lysates from cells transduced with a control shRNA (shCont) or with KRas shRNA (shKRas) lentiviruses using a western blot assay. (B) Serum-starved A549 shCont and shKRas cells were treated with EGF (100 ng/mL) for 0-60 min. Cell lysates were analyzed by western blot assay ( n = 3) with antibodies to pAkt, pERK1/2 and β-actin. (C) Serum-starved A549 or H1299 cells were treated with CBP501 for 24 h using transwell assay ( n = 3). (D) Serum-starved H1299-shCont or H1299-shKRas cells were treated with EGF (500 ng/mL) and IGF-I (200 ng/mL) in combination with CBP501 for 72 h using transwell assay ( n = 3). Data, the mean ± SD; * and **, P
    Figure Legend Snippet: KRas knockdown cells attenuate inhibition of cell migration by CBP501 (A) Stable silencing of KRas in A549 and H1299 cells was assessed by comparing KRas expression in cell lysates from cells transduced with a control shRNA (shCont) or with KRas shRNA (shKRas) lentiviruses using a western blot assay. (B) Serum-starved A549 shCont and shKRas cells were treated with EGF (100 ng/mL) for 0-60 min. Cell lysates were analyzed by western blot assay ( n = 3) with antibodies to pAkt, pERK1/2 and β-actin. (C) Serum-starved A549 or H1299 cells were treated with CBP501 for 24 h using transwell assay ( n = 3). (D) Serum-starved H1299-shCont or H1299-shKRas cells were treated with EGF (500 ng/mL) and IGF-I (200 ng/mL) in combination with CBP501 for 72 h using transwell assay ( n = 3). Data, the mean ± SD; * and **, P

    Techniques Used: Inhibition, Migration, Expressing, Transduction, shRNA, Western Blot, Transwell Assay

    CBP501 prevents cell migration and invasion in A549 and H1299 cell lines Serum-starved A549 ( A , left panel) or H1299 ( B , left panel) cells were treated with CBP501 (0.25, 0.5 or 1 μM) for 24 h using a transwell assay. Serum-starved A549 (A, right panel) or H1299 (B, right panel) cells were treated with CBP501 (1 μM) for 48 h using matrigel invasion chamber. Cell migration and invasion %ages (above) were calculated from sample eluted from the membrane ( n = 3). Photomicrographs of the observed cell migration (below). (C) Quantification of H1299 cell invasion in spheroid invasion assays. Cells were aggregated into spheroids and then induced to invade the surrounding matrix for 11 days. The total area of the invading spheroid was calculated with Image-J software and taken to be a measure of cell invasion ( n = 3). Red signal threshold was set to capture the total structure. Scale bar is 500 μm. Data, the mean ± SD; * and **, P
    Figure Legend Snippet: CBP501 prevents cell migration and invasion in A549 and H1299 cell lines Serum-starved A549 ( A , left panel) or H1299 ( B , left panel) cells were treated with CBP501 (0.25, 0.5 or 1 μM) for 24 h using a transwell assay. Serum-starved A549 (A, right panel) or H1299 (B, right panel) cells were treated with CBP501 (1 μM) for 48 h using matrigel invasion chamber. Cell migration and invasion %ages (above) were calculated from sample eluted from the membrane ( n = 3). Photomicrographs of the observed cell migration (below). (C) Quantification of H1299 cell invasion in spheroid invasion assays. Cells were aggregated into spheroids and then induced to invade the surrounding matrix for 11 days. The total area of the invading spheroid was calculated with Image-J software and taken to be a measure of cell invasion ( n = 3). Red signal threshold was set to capture the total structure. Scale bar is 500 μm. Data, the mean ± SD; * and **, P

    Techniques Used: Migration, Transwell Assay, Software

    27) Product Images from "ZMPSTE24 defends against influenza and other pathogenic viruses"

    Article Title: ZMPSTE24 defends against influenza and other pathogenic viruses

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161270

    ZMPSTE24 protects against viral infection. (A) ZMPSTE24-FLAG was transfected with HA-tagged STING, IFITM1, IFITM2, or IFITM3 into HEK293 cells. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody and blotted with the indicated antibodies. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IP, immunoprecipitation; WB, Western blot. (B) A549 cells were stimulated with 5 U IFNβ for the designated times. IFITM1, IFITM2, IFITM3, ZMPSTE24, and control GAPDH mRNA levels were examined by real-time PCR (three independent experiments). Mean ± SD; *, P
    Figure Legend Snippet: ZMPSTE24 protects against viral infection. (A) ZMPSTE24-FLAG was transfected with HA-tagged STING, IFITM1, IFITM2, or IFITM3 into HEK293 cells. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody and blotted with the indicated antibodies. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IP, immunoprecipitation; WB, Western blot. (B) A549 cells were stimulated with 5 U IFNβ for the designated times. IFITM1, IFITM2, IFITM3, ZMPSTE24, and control GAPDH mRNA levels were examined by real-time PCR (three independent experiments). Mean ± SD; *, P

    Techniques Used: Infection, Transfection, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction

    28) Product Images from "Genome-Wide Profiling Reveals That Herbal Medicine Jinfukang-Induced Polyadenylation Alteration Is Involved in Anti-Lung Cancer Activity"

    Article Title: Genome-Wide Profiling Reveals That Herbal Medicine Jinfukang-Induced Polyadenylation Alteration Is Involved in Anti-Lung Cancer Activity

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2017/5326909

    Characterization and comparative analysis of APA sites in A549 cells treated with JFK. (a) Distribution of 3T-seq reads across the gene body. (b) Genomic locations of poly (A) sites. (c) Position-specific distributions of PAS signal hexamer for poly (A) sites. (d) Statistics of genes with various number of detected poly (A) sites.
    Figure Legend Snippet: Characterization and comparative analysis of APA sites in A549 cells treated with JFK. (a) Distribution of 3T-seq reads across the gene body. (b) Genomic locations of poly (A) sites. (c) Position-specific distributions of PAS signal hexamer for poly (A) sites. (d) Statistics of genes with various number of detected poly (A) sites.

    Techniques Used:

    APA sites variations in A549 cells treated with JFK compared to control. (a) Scatter diagram of genes with differential APA defined by CULI. (b) Composition of downregulated and upregulated genes in A549 cells treated with JFK. (c) TMEM123 transcript isoforms with alternative poly (A) sites in JFK treated (Blue) and untreated (Brown) A549 cells. (d)(A) Shortened 3′ UTR of TMEM123 mRNA was verified by qRT-PCR. (B) Verification of TMEM123 expression level by qRT-PCR. Values in (d) represent means ± SE of data from three independent experiments, ∗∗∗ P
    Figure Legend Snippet: APA sites variations in A549 cells treated with JFK compared to control. (a) Scatter diagram of genes with differential APA defined by CULI. (b) Composition of downregulated and upregulated genes in A549 cells treated with JFK. (c) TMEM123 transcript isoforms with alternative poly (A) sites in JFK treated (Blue) and untreated (Brown) A549 cells. (d)(A) Shortened 3′ UTR of TMEM123 mRNA was verified by qRT-PCR. (B) Verification of TMEM123 expression level by qRT-PCR. Values in (d) represent means ± SE of data from three independent experiments, ∗∗∗ P

    Techniques Used: Quantitative RT-PCR, Expressing

    Treatment with JFK induced cell death in A549 cells. (a) Inhibitory effect of JFK on viability of A549 cells. A549 cells were treated with JFK (30 μ g/ml, 45 μ g/ml, 60 μ g/ml, 90 μ g/ml, 120 μ g/ml, 150 μ g/ml, and 200 μ g/ml, resp.) for 24 h; the viable cells were determined by CCK8 assay. (b) Observation of A549 cells under optical microscope and fluorescence microscope stained with PI for detecting dead cells. Scale bar: 50 μ m. (c) A549 cells were treated with JFK, and the apoptotic progression was determined by flow cytometry. Values in (a) represent means ± SE of data from three independent experiments, ∗∗∗ P
    Figure Legend Snippet: Treatment with JFK induced cell death in A549 cells. (a) Inhibitory effect of JFK on viability of A549 cells. A549 cells were treated with JFK (30 μ g/ml, 45 μ g/ml, 60 μ g/ml, 90 μ g/ml, 120 μ g/ml, 150 μ g/ml, and 200 μ g/ml, resp.) for 24 h; the viable cells were determined by CCK8 assay. (b) Observation of A549 cells under optical microscope and fluorescence microscope stained with PI for detecting dead cells. Scale bar: 50 μ m. (c) A549 cells were treated with JFK, and the apoptotic progression was determined by flow cytometry. Values in (a) represent means ± SE of data from three independent experiments, ∗∗∗ P

    Techniques Used: CCK-8 Assay, Microscopy, Fluorescence, Staining, Flow Cytometry, Cytometry

    Identification of APA sites in A549 cells treated with JFK. (a) Gel electrophoresis of the 3T-seq library; (b) a genomic view of APA sites defined by 3Tseq in IGV genome browser. Blue track: JFK; Yellow track: Control.
    Figure Legend Snippet: Identification of APA sites in A549 cells treated with JFK. (a) Gel electrophoresis of the 3T-seq library; (b) a genomic view of APA sites defined by 3Tseq in IGV genome browser. Blue track: JFK; Yellow track: Control.

    Techniques Used: Nucleic Acid Electrophoresis

    29) Product Images from "Recurrent rhinovirus infections in a child with inherited MDA5 deficiency"

    Article Title: Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161759

    Loss of MDA5 function does not affect RSV replication, whereas affecting RSV-induced IFN-regulated transcripts. (A) Number of RSV reads in RNA-seq data during RSV infection. RSV-infected (MOI: 1) A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg). (B) Immunoblotting showing efficiencies of MDA5 protein expression, after transient transfection of A549 cells with the indicated siRNA, or without transfection (MOCK). Cells were either left uninfected or infected with RSV, as indicated. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C) “Response to type I interferon” genes (GO term GO0034340) versus all genes, from A. (D) Heatmap representation of RNA-seq data showing the expression change between MDA5 siRNA and nonspecific siRNA control of IFN-regulated gene over the course of RSV infection. Mean of triplicate expression ratios from triplicate infections were shown for each time point in A, C, and D. (E) RSV transcripts in primary nasal epithelial cells from the patient (open bar), parents (hatched bars), and two normal healthy controls (solid bars), normalized to father at 6 h, after RSV-GFP infection (MOI: 0.2). (F) Percent GFP + of gated live RSV-infected cells from (E). Data show means ± SD from four experiments (E and F). *, P
    Figure Legend Snippet: Loss of MDA5 function does not affect RSV replication, whereas affecting RSV-induced IFN-regulated transcripts. (A) Number of RSV reads in RNA-seq data during RSV infection. RSV-infected (MOI: 1) A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg). (B) Immunoblotting showing efficiencies of MDA5 protein expression, after transient transfection of A549 cells with the indicated siRNA, or without transfection (MOCK). Cells were either left uninfected or infected with RSV, as indicated. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C) “Response to type I interferon” genes (GO term GO0034340) versus all genes, from A. (D) Heatmap representation of RNA-seq data showing the expression change between MDA5 siRNA and nonspecific siRNA control of IFN-regulated gene over the course of RSV infection. Mean of triplicate expression ratios from triplicate infections were shown for each time point in A, C, and D. (E) RSV transcripts in primary nasal epithelial cells from the patient (open bar), parents (hatched bars), and two normal healthy controls (solid bars), normalized to father at 6 h, after RSV-GFP infection (MOI: 0.2). (F) Percent GFP + of gated live RSV-infected cells from (E). Data show means ± SD from four experiments (E and F). *, P

    Techniques Used: RNA Sequencing Assay, Infection, Transfection, Negative Control, Expressing

    Loss of MDA5 function does not affect replication of influenza virus or production of proinflammatory cytokines in respiratory epithelial cells. (A) Influenza (MOI: 0.1) transcripts, normalized to siNeg control at 24 h. Influenza-infected A549 cells were previously transfected with the indicated siRNA. (B) Immunoblotting showing efficiencies of MDA5, RIG-I, and MAVS protein expression, relative to HSP90 loading control, after transient transfection with the indicated siRNA into A549 cells. Transfected cells were left uninfected or infected with influenza strain A/Victoria/361/2011 (MOI: 0.5) for 24 h or treated with IFN-β (10 IU/ml) for 24 h. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C–F) Proinflammatory gene transcripts quantitated by qRT-PCR from A. Levels of IL-1α (C), IL-6 (D), IL-8 (E), and TNF (F) were normalized to β-actin and are shown relative to normalized levels at 8 h after infection. Data show means ± SD from six to seven experiments (A) and three independent experiments in (C–F). *, P
    Figure Legend Snippet: Loss of MDA5 function does not affect replication of influenza virus or production of proinflammatory cytokines in respiratory epithelial cells. (A) Influenza (MOI: 0.1) transcripts, normalized to siNeg control at 24 h. Influenza-infected A549 cells were previously transfected with the indicated siRNA. (B) Immunoblotting showing efficiencies of MDA5, RIG-I, and MAVS protein expression, relative to HSP90 loading control, after transient transfection with the indicated siRNA into A549 cells. Transfected cells were left uninfected or infected with influenza strain A/Victoria/361/2011 (MOI: 0.5) for 24 h or treated with IFN-β (10 IU/ml) for 24 h. 20 µg of lysates were run per lane. Shown is a representative experiment corresponding to A. (C–F) Proinflammatory gene transcripts quantitated by qRT-PCR from A. Levels of IL-1α (C), IL-6 (D), IL-8 (E), and TNF (F) were normalized to β-actin and are shown relative to normalized levels at 8 h after infection. Data show means ± SD from six to seven experiments (A) and three independent experiments in (C–F). *, P

    Techniques Used: Infection, Transfection, Expressing, Quantitative RT-PCR

    MDA5 effects on IFN response during HRV infection . (A) “Response to type I interferon” genes (GO term GO0034340) versus all genes. (B) Heatmap representation of RNA-seq data showing HRV-induced IFN-regulated gene transcripts (RPKM) over the course of infection with HRV-B14 (MOI: 1). Data in A show box-and-whisker plots from triplicate infections. A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg) and correspond to Fig. 4 F . Mean of triplicate expression ratios from triplicate infections were shown for each time point in B. *, P
    Figure Legend Snippet: MDA5 effects on IFN response during HRV infection . (A) “Response to type I interferon” genes (GO term GO0034340) versus all genes. (B) Heatmap representation of RNA-seq data showing HRV-induced IFN-regulated gene transcripts (RPKM) over the course of infection with HRV-B14 (MOI: 1). Data in A show box-and-whisker plots from triplicate infections. A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg) and correspond to Fig. 4 F . Mean of triplicate expression ratios from triplicate infections were shown for each time point in B. *, P

    Techniques Used: Infection, RNA Sequencing Assay, Whisker Assay, Transfection, Negative Control, Expressing

    Loss of MDA5 function results in increased replication of HRV in respiratory epithelial cells . (A) HRV transcripts, normalized to nonspecific siRNA negative (siNeg) control at 20 h. HRV-B14–infected (MOI: 1) A549 cells were previously transfected with the indicated siRNA. (B) Immunoblot showing efficiencies of MDA5 and RIG-I knockdown in A. Transfected cells were left uninfected or infected with HRV-B14 for 48 h. 120 µg of lysates were run per lane. (C) Similar to A, at 48 h after infection and two rounds of transient transfection with the indicated siRNA. (D) Immunoblot showing efficiencies of MAVS and MDA5 knockdown in C. (E) HRV transcripts and HRV simultaneously quantitated by infectious plaque assay, at 60 h after HRV-B14 infection. (F) Number of HRV reads in RNA-seq data during HRV-B14 infection. A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg). Mean of triplicate expression ratios from triplicate infections were shown for each time point. (G) HRV transcripts, normalized to nonspecific siRNA negative (siNeg) control at 48 h, HRV-A16–infected (MOI: 10). Human primary fibroblasts were previously transfected with the indicated siRNA. (H) Immunoblot showing efficiencies of MDA5 and MAVS knockdown in G, except that cells were treated overnight with IFN-α. Data show means ± SD from six (A); eight (C), four (E), and seven experiments (G). Representative experiments are shown in B, D, and H. *, P
    Figure Legend Snippet: Loss of MDA5 function results in increased replication of HRV in respiratory epithelial cells . (A) HRV transcripts, normalized to nonspecific siRNA negative (siNeg) control at 20 h. HRV-B14–infected (MOI: 1) A549 cells were previously transfected with the indicated siRNA. (B) Immunoblot showing efficiencies of MDA5 and RIG-I knockdown in A. Transfected cells were left uninfected or infected with HRV-B14 for 48 h. 120 µg of lysates were run per lane. (C) Similar to A, at 48 h after infection and two rounds of transient transfection with the indicated siRNA. (D) Immunoblot showing efficiencies of MAVS and MDA5 knockdown in C. (E) HRV transcripts and HRV simultaneously quantitated by infectious plaque assay, at 60 h after HRV-B14 infection. (F) Number of HRV reads in RNA-seq data during HRV-B14 infection. A549 cells were previously transfected with MDA5 or nonspecific negative control siRNA (siNeg). Mean of triplicate expression ratios from triplicate infections were shown for each time point. (G) HRV transcripts, normalized to nonspecific siRNA negative (siNeg) control at 48 h, HRV-A16–infected (MOI: 10). Human primary fibroblasts were previously transfected with the indicated siRNA. (H) Immunoblot showing efficiencies of MDA5 and MAVS knockdown in G, except that cells were treated overnight with IFN-α. Data show means ± SD from six (A); eight (C), four (E), and seven experiments (G). Representative experiments are shown in B, D, and H. *, P

    Techniques Used: Infection, Transfection, Plaque Assay, RNA Sequencing Assay, Negative Control, Expressing

    Loss of MDA5 function results in increased replication of HRV in respiratory epithelial cells and fibroblasts . (A) HRV transcripts, normalized to father control at 48 h. Primary nasal epithelial cells from the patient, parents, and six normal healthy controls were infected with HRV-B14 (MOI: 1) as indicated. (B) HRV transcripts in maternal fibroblasts, gene-edited to have IFIH1 genotypes WT/- (2 clones), WT/K365E (1 clone), K365E/- (2 clones), −/− (3 clones), after infection with HRV-B14 (MOI: 1). (C) HRV transcripts in A549 cells previously transduced with empty vector (EV), WT, or K365E MDA5, at 72 h after infection with HRV-B14 (MOI:1). (D) Immunoblot showing MDA5 overexpression and comparable RIG-Iexpression in C. 10.5 µg of lysates were run per lane. Data show means ± SD from two (A), six (B), and five experiments (C). *, P
    Figure Legend Snippet: Loss of MDA5 function results in increased replication of HRV in respiratory epithelial cells and fibroblasts . (A) HRV transcripts, normalized to father control at 48 h. Primary nasal epithelial cells from the patient, parents, and six normal healthy controls were infected with HRV-B14 (MOI: 1) as indicated. (B) HRV transcripts in maternal fibroblasts, gene-edited to have IFIH1 genotypes WT/- (2 clones), WT/K365E (1 clone), K365E/- (2 clones), −/− (3 clones), after infection with HRV-B14 (MOI: 1). (C) HRV transcripts in A549 cells previously transduced with empty vector (EV), WT, or K365E MDA5, at 72 h after infection with HRV-B14 (MOI:1). (D) Immunoblot showing MDA5 overexpression and comparable RIG-Iexpression in C. 10.5 µg of lysates were run per lane. Data show means ± SD from two (A), six (B), and five experiments (C). *, P

    Techniques Used: Infection, Transduction, Plasmid Preparation, Over Expression

    30) Product Images from "Tumor-suppressive miR-218-5p inhibits cancer cell proliferation and migration via EGFR in non-small cell lung cancer"

    Article Title: Tumor-suppressive miR-218-5p inhibits cancer cell proliferation and migration via EGFR in non-small cell lung cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8576

    miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the β-galactosidase levels of the control (**P
    Figure Legend Snippet: miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the β-galactosidase levels of the control (**P

    Techniques Used: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Transfection

    miR-218-5p inhibits the migration and proliferation of NSCLC cells in vitro A and B. Transwell analyses of the migrated A549 (A) and H1975 (B) cells transfected with the miR-218-5p mimic, scrambled mimic, antisense miR-218-5p or scrambled antisense miRNA. A549 cells were allowed to migrate for 6 h and H1975 cells were allowed to migrate for 12 h. Left panel: representative images showing cells that migrated through the filter; right panel: cell numbers counted in each field (*P
    Figure Legend Snippet: miR-218-5p inhibits the migration and proliferation of NSCLC cells in vitro A and B. Transwell analyses of the migrated A549 (A) and H1975 (B) cells transfected with the miR-218-5p mimic, scrambled mimic, antisense miR-218-5p or scrambled antisense miRNA. A549 cells were allowed to migrate for 6 h and H1975 cells were allowed to migrate for 12 h. Left panel: representative images showing cells that migrated through the filter; right panel: cell numbers counted in each field (*P

    Techniques Used: Migration, In Vitro, Transfection

    Effects of EGFR knockdown on the proliferation of NSCLC cells A. Western blotting analyses of the expression levels of the EGFR protein in the A549 and H1975 cells transfected with scrambled siRNA or EGFR siRNAs (EGFR siRNA-1 and EGFR siRNA-2). Upper panel: representative images; lower panel: quantitative analysis (**P
    Figure Legend Snippet: Effects of EGFR knockdown on the proliferation of NSCLC cells A. Western blotting analyses of the expression levels of the EGFR protein in the A549 and H1975 cells transfected with scrambled siRNA or EGFR siRNAs (EGFR siRNA-1 and EGFR siRNA-2). Upper panel: representative images; lower panel: quantitative analysis (**P

    Techniques Used: Western Blot, Expressing, Transfection

    31) Product Images from "Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells"

    Article Title: Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12647

    Methylation status of RBL1 promotor after X-rays or 5-Aza-CdR treatment and RBL1 expression in 3D A549 cells A . The changes of promoter methylation status of RBL1 at indicated time points measured by MSP in 4 Gy X-ray irradiated 2D and 3D cells. B . The changes of promoter methylation status of RBL1 measured by MSP in 5-Aza-CdR treated 2D and 3D cells. C . The expression of RBL1 in 2D and 3D cells at indicated time points after irradiation with 4 Gy X-rays by Western blot assay. D . The expression of RBL1 in 5-Aza-CdR treated 2D and 3D A549 cells by Western blot assay. E F . Cell cycle distribution of 2D and 3D cells after 4 Gy X-ray irradiation combined with 2 μM 5-Aza-CdR treatment. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: Methylation status of RBL1 promotor after X-rays or 5-Aza-CdR treatment and RBL1 expression in 3D A549 cells A . The changes of promoter methylation status of RBL1 at indicated time points measured by MSP in 4 Gy X-ray irradiated 2D and 3D cells. B . The changes of promoter methylation status of RBL1 measured by MSP in 5-Aza-CdR treated 2D and 3D cells. C . The expression of RBL1 in 2D and 3D cells at indicated time points after irradiation with 4 Gy X-rays by Western blot assay. D . The expression of RBL1 in 5-Aza-CdR treated 2D and 3D A549 cells by Western blot assay. E F . Cell cycle distribution of 2D and 3D cells after 4 Gy X-ray irradiation combined with 2 μM 5-Aza-CdR treatment. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Methylation, Expressing, Irradiation, Western Blot

    Overexpression of RBL1 sensitizes the 3D cultured A549 and MCF7 cells to X-rays A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 3D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 3D A549 and MCF7 cells. C D . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 3D A549 and MCF7 cells. F . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 3D A549 and MCF7 cells. G H . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1-siRNA or NC, were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: Overexpression of RBL1 sensitizes the 3D cultured A549 and MCF7 cells to X-rays A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 3D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 3D A549 and MCF7 cells. C D . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 3D A549 and MCF7 cells. F . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 3D A549 and MCF7 cells. G H . Survival fractions of 3D A549 and MCF7 cells transfected with RBL1-siRNA or NC, were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Over Expression, Cell Culture, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Colony Assay, Negative Control

    The 3D cultured cells are radioresistant compared with 2D cultured cells A B . Survival fraction of A549 and MCF7 cells cultured in 2D and 3D after exposed to 0, 1, 2, 4 and 6 Gy X-rays and measured by colony formation assay. C . Micronucleus frequency of A549 and MCF7 cells cultured in 2D and 3D after exposed to 0, 1, 2, 4 and 6 Gy X-rays. D E . Cell cycle distribution of 2D and 3D A549 cells after exposure to 4 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: The 3D cultured cells are radioresistant compared with 2D cultured cells A B . Survival fraction of A549 and MCF7 cells cultured in 2D and 3D after exposed to 0, 1, 2, 4 and 6 Gy X-rays and measured by colony formation assay. C . Micronucleus frequency of A549 and MCF7 cells cultured in 2D and 3D after exposed to 0, 1, 2, 4 and 6 Gy X-rays. D E . Cell cycle distribution of 2D and 3D A549 cells after exposure to 4 Gy X-rays. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Cell Culture, Colony Assay

    Expression of cell cycle regulation genes in irradiated 2D and 3D A549 cells A . Microarray analysis of 84 cell cycle regulation genes expression profile in 2D and 3D A549 cells 12h after irradiation with 4Gy X-rays. B . Venn diagrams of different expression of 84 cell cycle genes. C . Relative expression levels of RBL1, CCND1 and CCNF were measured by qRT-PCR at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells. GAPDH were used as internal control. D . The expressions of RBL1, CCND1 and CCNF at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells by western blot assay. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: Expression of cell cycle regulation genes in irradiated 2D and 3D A549 cells A . Microarray analysis of 84 cell cycle regulation genes expression profile in 2D and 3D A549 cells 12h after irradiation with 4Gy X-rays. B . Venn diagrams of different expression of 84 cell cycle genes. C . Relative expression levels of RBL1, CCND1 and CCNF were measured by qRT-PCR at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells. GAPDH were used as internal control. D . The expressions of RBL1, CCND1 and CCNF at the indicated time points after 4 Gy X-rays in 2D and 3D A549 cells by western blot assay. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Expressing, Irradiation, Microarray, Quantitative RT-PCR, Western Blot

    Methylation of genome and cell cycle regulation genes promotor status in irradiated 2D and 3D A549 cells A . Global DNA methylation status at the indicated 12h time points after 4 Gy X-rays irradiation in 2D and 3D cells. B C . Methylation PCR array of 22 cell cycle genes promotor at 12 h after irradiation with 4 Gy X-rays in 2D and 3D cells. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: Methylation of genome and cell cycle regulation genes promotor status in irradiated 2D and 3D A549 cells A . Global DNA methylation status at the indicated 12h time points after 4 Gy X-rays irradiation in 2D and 3D cells. B C . Methylation PCR array of 22 cell cycle genes promotor at 12 h after irradiation with 4 Gy X-rays in 2D and 3D cells. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Methylation, Irradiation, DNA Methylation Assay, Polymerase Chain Reaction

    Knockdown of RBL1 enhances the radioresistance of 2D cultured A549 and MCF7 cells and decreases the G2/M arrest induced by X-ray A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 2D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 2D A549 and MCF7 cells. C D . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1-siRNA or NC, was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E F . Cell cycle distribution of 2D A549 cells transfected with RBL1-siRNA or NC and then exposed 4 Gy X-rays. G . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 2D A549 and MCF7 cells. H . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 2D A549 and MCF7 cells. I J . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P
    Figure Legend Snippet: Knockdown of RBL1 enhances the radioresistance of 2D cultured A549 and MCF7 cells and decreases the G2/M arrest induced by X-ray A . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after transfected with RBL1-siRNA or negative control (NC) in 2D A549 and MCF7 cells. GAPDH were used as internal control. B . Western blot assay of RBL1 protein level after being transfected with RBL1-siRNA or NC into 2D A549 and MCF7 cells. C D . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1-siRNA or NC, was measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. E F . Cell cycle distribution of 2D A549 cells transfected with RBL1-siRNA or NC and then exposed 4 Gy X-rays. G . Relative expression levels of RBL1 mRNA were measured by qRT-PCR after being transfected with RBL1 overexpression vector or negative vector (NC) in 2D A549 and MCF7 cells. H . Western blot assay of RBL1 protein level after being transfected with RBL1 overexpression vector or NC into 2D A549 and MCF7 cells. I J . Survival fractions of 2D A549 and MCF7 cells transfected with RBL1 overexpression vector or NC were measured by colony formation assay after exposed to 0, 1, 2, 4 and 6 Gy X-rays. Each data point represents the mean of three separate experiments. Bars are the standard errors. Significance was determined by Student's t-test. *, P

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Colony Assay, Over Expression, Plasmid Preparation

    Different morphological features and biomarker expressions of organotypic tissue between 2D and 3D cultured cells The morphology of 2D and 3D cultured A549 and MCF7 cells were captured under phase-contrast microscope A . The 2D and 3D cultured A549 cells were immunostained with anti-Cytokeratin 5 B . anti-pan-Cytokeratin C .,anti-ZO-1 D . and anti-β-tublin E . and the representative confocal images are shown. F . Fold changes ofCytokeratin 2, 5 and ZO-1 mRNAs in 2D and 3D A549 cells. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. **, P
    Figure Legend Snippet: Different morphological features and biomarker expressions of organotypic tissue between 2D and 3D cultured cells The morphology of 2D and 3D cultured A549 and MCF7 cells were captured under phase-contrast microscope A . The 2D and 3D cultured A549 cells were immunostained with anti-Cytokeratin 5 B . anti-pan-Cytokeratin C .,anti-ZO-1 D . and anti-β-tublin E . and the representative confocal images are shown. F . Fold changes ofCytokeratin 2, 5 and ZO-1 mRNAs in 2D and 3D A549 cells. Each data point represents the mean of three independent experiments. Bars are the standard errors. Significance was determined by Student's t-test. **, P

    Techniques Used: Biomarker Assay, Cell Culture, Microscopy

    32) Product Images from "Orally active microtubule-targeting agent, MPT0B271, for the treatment of human non-small cell lung cancer, alone and in combination with erlotinib"

    Article Title: Orally active microtubule-targeting agent, MPT0B271, for the treatment of human non-small cell lung cancer, alone and in combination with erlotinib

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.128

    ( a ) Measurement of apoptosis. A549 cells were treated with the indicated concentration of MPT0B271, and oligonucleosomal DNA fragmentation was quantitatively assessed with the Cell Death ELISA PLUS kit. Apoptosis was enhanced in relation to control cells. Data are expressed as the mean±S.E. of at least three independent experiments. *** P
    Figure Legend Snippet: ( a ) Measurement of apoptosis. A549 cells were treated with the indicated concentration of MPT0B271, and oligonucleosomal DNA fragmentation was quantitatively assessed with the Cell Death ELISA PLUS kit. Apoptosis was enhanced in relation to control cells. Data are expressed as the mean±S.E. of at least three independent experiments. *** P

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    ( a ) The effect of MPT0B271 on p53 expression and phosphorylation in A549 cells. Cells were treated with MPT0B271 (0.3 μ M) for the indicated times, and whole-cell extracts were prepared and underwent western blot analysis using the indicated antibodies. ( b ) A549 cells were pretreated with or without p53 siRNA for 24 h and then incubated with or without MPT0B271 (0.1 μ M) for 48 h. Upper panel , total cellular lysates were subjected to western blot analysis of p53 and PARP. Lower panel , cell viability was measured by the SRB assay and expressed as a percentage of the untreated control. ( c ) Concentration-dependent effect of MPT0B271 on cell viability. H1299 and H226 cells were treated with or without the indicated concentration of MPT0B271 for 48 h, and the cytotoxic effect was determined with the MTT assay. Data are expressed as the mean±S.E. of at least three independent experiments. * P
    Figure Legend Snippet: ( a ) The effect of MPT0B271 on p53 expression and phosphorylation in A549 cells. Cells were treated with MPT0B271 (0.3 μ M) for the indicated times, and whole-cell extracts were prepared and underwent western blot analysis using the indicated antibodies. ( b ) A549 cells were pretreated with or without p53 siRNA for 24 h and then incubated with or without MPT0B271 (0.1 μ M) for 48 h. Upper panel , total cellular lysates were subjected to western blot analysis of p53 and PARP. Lower panel , cell viability was measured by the SRB assay and expressed as a percentage of the untreated control. ( c ) Concentration-dependent effect of MPT0B271 on cell viability. H1299 and H226 cells were treated with or without the indicated concentration of MPT0B271 for 48 h, and the cytotoxic effect was determined with the MTT assay. Data are expressed as the mean±S.E. of at least three independent experiments. * P

    Techniques Used: Expressing, Western Blot, Incubation, Sulforhodamine B Assay, Concentration Assay, MTT Assay

    ( a ) Effect of MPT0B271 on cell-cycle progression. A549 cells were exposed to 0.3 μ M MPT0B271 for the indicated times and then stained with PI to determine the proportion of DNA. Data acquisition and analysis were performed on a FACScan flow cytometer. The data are expressed as the mean±S.E. of at least three independent experiments. ( b ) The effect of MPT0B271 on G2/M cell-cycle regulatory proteins. A549 cells were treated with 0.3 μ M MPT0B271 for the indicated times. Whole-cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) Treatment with 0.3 μ M MPT0B271 for the indicated times. Nuclear lysates were subjected to western blot analysis using an antibody specific for cyclin B1
    Figure Legend Snippet: ( a ) Effect of MPT0B271 on cell-cycle progression. A549 cells were exposed to 0.3 μ M MPT0B271 for the indicated times and then stained with PI to determine the proportion of DNA. Data acquisition and analysis were performed on a FACScan flow cytometer. The data are expressed as the mean±S.E. of at least three independent experiments. ( b ) The effect of MPT0B271 on G2/M cell-cycle regulatory proteins. A549 cells were treated with 0.3 μ M MPT0B271 for the indicated times. Whole-cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) Treatment with 0.3 μ M MPT0B271 for the indicated times. Nuclear lysates were subjected to western blot analysis using an antibody specific for cyclin B1

    Techniques Used: Staining, Flow Cytometry, Cytometry, SDS Page, Western Blot

    ( a ) Chemical structure of MPT0B271. ( b ) Effect of MPT0B271 on tubulin polymerization. Purified tubulin in reaction buffer was incubated at 37°C in the absence or presence of increasing concentrations of MPT0B271, 10 μ M paclitaxel, 10 μ M colchicine, or 10 μ M vincristine. Assembly of microtubules was then measured over 30 min at 1 min intervals at an absorbance of 340 nm using a spectrophotometer. ( c ) Immunofluorescence staining of microtubules in A549 cells. Cells were treated with vehicle (DMSO), 0.3 μ M MPT0B271, paclitaxel, or vincristine for 24 h. Cells were labeled with a β -tubulin antibody and an FITC-conjugated anti-mouse IgG antibody, were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and observed by confocal microscopy. Left , DAPI; middle , microtubule network; right , merged microtubule network and DAPI. ( d ) PK properties, plasma concentration versus time profiles of MPT0B271 after i.v. (2 mg/kg) and p.o. (20 mg/kg) dosing of fasted male CD-1 ( Crl. ) mice. ( e ) Efficacy of MPT0B271, dosed orally, on tumor xenografts. Upper panel , tumor growth of A549 xenografts in nude mice that were orally treated with or without MPT0B271 (5, 10, and 20 mg/kg). Tumor growth is presented as the mean tumor volume (mm 3 )±S.E. Tumor volume was determined using caliper measurements and was calculated as the product of 1/2 × length × width 2 . Lower panel , body weight (g) of the mice. * P
    Figure Legend Snippet: ( a ) Chemical structure of MPT0B271. ( b ) Effect of MPT0B271 on tubulin polymerization. Purified tubulin in reaction buffer was incubated at 37°C in the absence or presence of increasing concentrations of MPT0B271, 10 μ M paclitaxel, 10 μ M colchicine, or 10 μ M vincristine. Assembly of microtubules was then measured over 30 min at 1 min intervals at an absorbance of 340 nm using a spectrophotometer. ( c ) Immunofluorescence staining of microtubules in A549 cells. Cells were treated with vehicle (DMSO), 0.3 μ M MPT0B271, paclitaxel, or vincristine for 24 h. Cells were labeled with a β -tubulin antibody and an FITC-conjugated anti-mouse IgG antibody, were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and observed by confocal microscopy. Left , DAPI; middle , microtubule network; right , merged microtubule network and DAPI. ( d ) PK properties, plasma concentration versus time profiles of MPT0B271 after i.v. (2 mg/kg) and p.o. (20 mg/kg) dosing of fasted male CD-1 ( Crl. ) mice. ( e ) Efficacy of MPT0B271, dosed orally, on tumor xenografts. Upper panel , tumor growth of A549 xenografts in nude mice that were orally treated with or without MPT0B271 (5, 10, and 20 mg/kg). Tumor growth is presented as the mean tumor volume (mm 3 )±S.E. Tumor volume was determined using caliper measurements and was calculated as the product of 1/2 × length × width 2 . Lower panel , body weight (g) of the mice. * P

    Techniques Used: Purification, Incubation, Spectrophotometry, Immunofluorescence, Staining, Labeling, Confocal Microscopy, Concentration Assay, Mouse Assay

    In vitro and in vivo antitumor activity of MPT0B271 in combination with erlotinib. ( a ) A549 cells were treated with erlotinib (5 μ M) in combination with MPT0B271 (0.0125 or 0.025 μ M) for 48 h, and cell apoptosis was measured using the Cell Death ELISA PLUS kit. Data are expressed as the mean±S.E. of at least three independent determinations. ( b ) A549 xenograft model. A549-tumor-bearing nude mice were treated with vehicle, MPT0B271 (20 mg/kg/day by oral gavage q2d), erlotinib (25 mg/kg/day by oral gavage once a day), or MPT0B271 in combination with erlotinib. Tumor was excised when the tumor size reached 1200 mm 3 . ( c ) The body weight of the mice measured daily during the first week and then at the days of administration. * P
    Figure Legend Snippet: In vitro and in vivo antitumor activity of MPT0B271 in combination with erlotinib. ( a ) A549 cells were treated with erlotinib (5 μ M) in combination with MPT0B271 (0.0125 or 0.025 μ M) for 48 h, and cell apoptosis was measured using the Cell Death ELISA PLUS kit. Data are expressed as the mean±S.E. of at least three independent determinations. ( b ) A549 xenograft model. A549-tumor-bearing nude mice were treated with vehicle, MPT0B271 (20 mg/kg/day by oral gavage q2d), erlotinib (25 mg/kg/day by oral gavage once a day), or MPT0B271 in combination with erlotinib. Tumor was excised when the tumor size reached 1200 mm 3 . ( c ) The body weight of the mice measured daily during the first week and then at the days of administration. * P

    Techniques Used: In Vitro, In Vivo, Activity Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

    33) Product Images from "Gambogic acid suppresses cancer invasion and migration by inhibiting TGFβ1-induced epithelial-to-mesenchymal transition"

    Article Title: Gambogic acid suppresses cancer invasion and migration by inhibiting TGFβ1-induced epithelial-to-mesenchymal transition

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15449

    GA inhibits TGFβ1+TNFα-activated NF-κB signaling in A549 cells (A) A549 cells were treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. Western blotting analyses of p-IKKα, IKKα, p-IκBα, and IκBα were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. ( B ) Cytosolic fractions and nuclear extracts were prepared from cells treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. Western blotting analysis of NF-κB p65 was performed to investigate the nuclear translocation of NF-κB p65. (C) A549 cells were transiently transfected with an NF-κB reporter gene plasmid for 5 h. After transfection, the cells were washed and treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. The luciferase activities were detected as described in the Materials and methods. Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA inhibits TGFβ1+TNFα-activated NF-κB signaling in A549 cells (A) A549 cells were treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. Western blotting analyses of p-IKKα, IKKα, p-IκBα, and IκBα were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. ( B ) Cytosolic fractions and nuclear extracts were prepared from cells treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. Western blotting analysis of NF-κB p65 was performed to investigate the nuclear translocation of NF-κB p65. (C) A549 cells were transiently transfected with an NF-κB reporter gene plasmid for 5 h. After transfection, the cells were washed and treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. The luciferase activities were detected as described in the Materials and methods. Each experiment was performed at least three times. *p

    Techniques Used: Western Blot, Translocation Assay, Transfection, Plasmid Preparation, Luciferase

    GA suppresses lung growth and metastasis of A549 cells in vivo Murine lung orthotopic model of A549 cells was conducted to determine the effect of GA in vivo . (A) Macroscopic lung lesions were detected. After the lungs were fixed in Bouin's solution, the numbers of lung metastatic nodules on the lung surfaces were quantified. (B) H E stained bilateral lungs of mice from GA-treated and control group to confirm the malignance of nodules were and the presence of micrometastases. Each bar represents 100 μm. (C) Immunohistochemical detection of E-cadherin, MMP-9 and vimentin protein levels in the right lung (100×). Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA suppresses lung growth and metastasis of A549 cells in vivo Murine lung orthotopic model of A549 cells was conducted to determine the effect of GA in vivo . (A) Macroscopic lung lesions were detected. After the lungs were fixed in Bouin's solution, the numbers of lung metastatic nodules on the lung surfaces were quantified. (B) H E stained bilateral lungs of mice from GA-treated and control group to confirm the malignance of nodules were and the presence of micrometastases. Each bar represents 100 μm. (C) Immunohistochemical detection of E-cadherin, MMP-9 and vimentin protein levels in the right lung (100×). Each experiment was performed at least three times. *p

    Techniques Used: In Vivo, Staining, Mouse Assay, Immunohistochemistry

    Inhibition of TGFβ1-induced EMT by GA does not require SMAD pathway or the transferrin receptor A549 cells were treated with the indicated concentrations of GA, TGFβ1 and SIS3 for 24 h. (A) Cytosolic fractions and nuclear extracts were prepared. Western blotting analysis of total SMAD4 and the nuclear translocation of SMAD4 were performed with specific antibodies. (B) Western blotting analyses of p-SMAD3 and SMAD3 were performed with whole-cell lysates and specific antibodies. (C) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-SMAD3 and SMAD3 were performed with whole-cell lysates and specific antibodies. (D) A549 cells were transfected with transferrin receptor siRNA and scrambled siRNA, and TfR mRNA level is used to measure transfection efficiency. When cells are transfected at efficiencies of 50–60%, western blotting analyses of transferrin receptor, E-cadherin, vimentin, TWIST1 were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p
    Figure Legend Snippet: Inhibition of TGFβ1-induced EMT by GA does not require SMAD pathway or the transferrin receptor A549 cells were treated with the indicated concentrations of GA, TGFβ1 and SIS3 for 24 h. (A) Cytosolic fractions and nuclear extracts were prepared. Western blotting analysis of total SMAD4 and the nuclear translocation of SMAD4 were performed with specific antibodies. (B) Western blotting analyses of p-SMAD3 and SMAD3 were performed with whole-cell lysates and specific antibodies. (C) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-SMAD3 and SMAD3 were performed with whole-cell lysates and specific antibodies. (D) A549 cells were transfected with transferrin receptor siRNA and scrambled siRNA, and TfR mRNA level is used to measure transfection efficiency. When cells are transfected at efficiencies of 50–60%, western blotting analyses of transferrin receptor, E-cadherin, vimentin, TWIST1 were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p

    Techniques Used: Inhibition, Western Blot, Translocation Assay, Transfection

    GA regulates the expression of EMT-related biomarkers and transcription factor TWIST1 A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. (A) A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. The expression of E-cadherin, vimentin, N-cadherin, MMP9, and RAC1 proteins in the cells was analyzed by western blotting using specific antibodies. An anti-β-actin antibody was used to check equivalent protein loading. (B) The expression of transcription factors TWIST1 and SNAIL was measured with western blotting. The values under each lane indicate the relative densities of the bands normalized to that of β-actin. (C) E-cadherin, vimentin, N-cadherin, MMP9, RAC1 and TWIST1 and SNAIL mRNAs were measured with real-time PCR. β-Actin was used as the internal control. The relative levels were calculated as the ratio of the relative biomarker mRNA to β-actin mRNA. Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA regulates the expression of EMT-related biomarkers and transcription factor TWIST1 A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. (A) A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. The expression of E-cadherin, vimentin, N-cadherin, MMP9, and RAC1 proteins in the cells was analyzed by western blotting using specific antibodies. An anti-β-actin antibody was used to check equivalent protein loading. (B) The expression of transcription factors TWIST1 and SNAIL was measured with western blotting. The values under each lane indicate the relative densities of the bands normalized to that of β-actin. (C) E-cadherin, vimentin, N-cadherin, MMP9, RAC1 and TWIST1 and SNAIL mRNAs were measured with real-time PCR. β-Actin was used as the internal control. The relative levels were calculated as the ratio of the relative biomarker mRNA to β-actin mRNA. Each experiment was performed at least three times. *p

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Biomarker Assay

    GA inhibits TGFβ1-induced activation of the NF-κB pathways in A549 cells A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. (A) Western blotting analyses of p-IKKα, IKKα, p-IκBα, and IκBα were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. (B) Nuclear extracts wereprepared and assayed for NF-κB activation with EMSA, using 8 μg of nuclear extract. (C) Immunoprecipitated DNA was extracted and dissolved and then DNA was measured by RT-PCR. The relative levels were calculated as the ratio of TWIST to β-actin mRNA expression, compared with the Input. Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA inhibits TGFβ1-induced activation of the NF-κB pathways in A549 cells A549 cells were treated with the indicated concentrations of GA and TGFβ1 for 24 h. (A) Western blotting analyses of p-IKKα, IKKα, p-IκBα, and IκBα were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. (B) Nuclear extracts wereprepared and assayed for NF-κB activation with EMSA, using 8 μg of nuclear extract. (C) Immunoprecipitated DNA was extracted and dissolved and then DNA was measured by RT-PCR. The relative levels were calculated as the ratio of TWIST to β-actin mRNA expression, compared with the Input. Each experiment was performed at least three times. *p

    Techniques Used: Activation Assay, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing

    GA inhibits TGFβ1+TNFα-induced EMT and the invasion of A549 cells A549 cells were treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. (A) An MTT assay showed that GA, TGFβ1, and TNFα had no effect on cell viability. (B) Morphological observation of A549 cells in the presence of the indicated concentrations of GA, TGFβ1, and TNFα with microscopy (image magnification: 100×). (C) An in vitro cell invasion assay showed that GA inhibits TGFβ1+TNFα-induced cell invasion (image magnification: 200×). (D) The expression of E-cadherin, vimentin, and TWIST1 proteins in the cells was analyzed with western blotting using specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA inhibits TGFβ1+TNFα-induced EMT and the invasion of A549 cells A549 cells were treated with the indicated concentrations of GA, TGFβ1, and TNFα for 24 h. (A) An MTT assay showed that GA, TGFβ1, and TNFα had no effect on cell viability. (B) Morphological observation of A549 cells in the presence of the indicated concentrations of GA, TGFβ1, and TNFα with microscopy (image magnification: 100×). (C) An in vitro cell invasion assay showed that GA inhibits TGFβ1+TNFα-induced cell invasion (image magnification: 200×). (D) The expression of E-cadherin, vimentin, and TWIST1 proteins in the cells was analyzed with western blotting using specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p

    Techniques Used: MTT Assay, Microscopy, In Vitro, Invasion Assay, Expressing, Western Blot

    Inhibition of TGFβ1-induced EMT by GA does not require MAPK pathway A549 cells were treated with the indicated concentrations of GA, TGFβ1, U0126 and SB203580 for 24 h. (A) Western blotting analyses of p-MEK, MEK1/2, p-p38 and p38 were performed with whole-cell lysates and specific antibodies. (B) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-MEK and MEK1/2 were performed with whole-cell lysates and specific antibodies. (C) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-p38 and p38 were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p
    Figure Legend Snippet: Inhibition of TGFβ1-induced EMT by GA does not require MAPK pathway A549 cells were treated with the indicated concentrations of GA, TGFβ1, U0126 and SB203580 for 24 h. (A) Western blotting analyses of p-MEK, MEK1/2, p-p38 and p38 were performed with whole-cell lysates and specific antibodies. (B) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-MEK and MEK1/2 were performed with whole-cell lysates and specific antibodies. (C) Western blotting analyses of E-cadherin, vimentin, TWIST1, p-p38 and p38 were performed with whole-cell lysates and specific antibodies. Anti-β-actin antibody was used to confirm equivalent protein loading. Each experiment was performed at least three times. *p

    Techniques Used: Inhibition, Western Blot

    GA inhibits TGFβ1-induced migration and invasion of A549 cells in vitro (A) Cells were exposed to different concentrations of GA and TGFβ1 for 24 h in a 5% CO 2 incubator at 37°C. An MTT assay then showed that GA and TGFβ1 had no effect on cell viability. (B) A monolayer of A549 cells was scraped with a pipette tip and then treated with different concentrations of GA and TGFβ1 for 12 or 24 h. The migrating cells were assessed with a microscope equipped with a camera. The results show that GA inhibits TGFβ1-induced cell migration (image magnification: 100×). (C) After A549 cells were treated with different concentrations of GA and TGFβ1 for 24 h, their invasive ability was evaluated with a Matrigel-based in vitro invasion assay. GA inhibited TGFβ1-induced cell invasion (image magnification: 200×). (D) A549 cells were treated with different concentrations of GA and TGFβ1 for 24 h, and a FITC–phalloidin staining assay was performed to observe the structure of F-actin with confocal microscopy (image magnification: 400×). Each experiment was performed at least three times. *p
    Figure Legend Snippet: GA inhibits TGFβ1-induced migration and invasion of A549 cells in vitro (A) Cells were exposed to different concentrations of GA and TGFβ1 for 24 h in a 5% CO 2 incubator at 37°C. An MTT assay then showed that GA and TGFβ1 had no effect on cell viability. (B) A monolayer of A549 cells was scraped with a pipette tip and then treated with different concentrations of GA and TGFβ1 for 12 or 24 h. The migrating cells were assessed with a microscope equipped with a camera. The results show that GA inhibits TGFβ1-induced cell migration (image magnification: 100×). (C) After A549 cells were treated with different concentrations of GA and TGFβ1 for 24 h, their invasive ability was evaluated with a Matrigel-based in vitro invasion assay. GA inhibited TGFβ1-induced cell invasion (image magnification: 200×). (D) A549 cells were treated with different concentrations of GA and TGFβ1 for 24 h, and a FITC–phalloidin staining assay was performed to observe the structure of F-actin with confocal microscopy (image magnification: 400×). Each experiment was performed at least three times. *p

    Techniques Used: Migration, In Vitro, MTT Assay, Transferring, Microscopy, Invasion Assay, Staining, Confocal Microscopy

    34) Product Images from "Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress"

    Article Title: Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18050966

    Plasma induced cell death in an energy dependent manner. ( A ) 1 h after plasma exposure, A549 viability decreased as quantified with a propidium iodide (PI) exclusion assay, indicating early damaging effects of plasma (One Way ANOVA, Dunnett’s multiple comparison test). ( B ) Representative dot plots of Annexin V/PI double stained cells 24 h post plasma treatment. Results are summarized in ( C ). Early apoptotic cells (Annexin V+/PI−) were increased significantly compared to untreated controls for all energies above 50 mJ (Two Way ANOVA, Sidak’s multiple comparison test). Data are represented as mean ± SEM. ** p
    Figure Legend Snippet: Plasma induced cell death in an energy dependent manner. ( A ) 1 h after plasma exposure, A549 viability decreased as quantified with a propidium iodide (PI) exclusion assay, indicating early damaging effects of plasma (One Way ANOVA, Dunnett’s multiple comparison test). ( B ) Representative dot plots of Annexin V/PI double stained cells 24 h post plasma treatment. Results are summarized in ( C ). Early apoptotic cells (Annexin V+/PI−) were increased significantly compared to untreated controls for all energies above 50 mJ (Two Way ANOVA, Sidak’s multiple comparison test). Data are represented as mean ± SEM. ** p

    Techniques Used: Exclusion Assay, Staining

    Plasma treatment of tumor cells in immunogenic cell death (ICD)-inducing regimes enhanced anti-tumor activity of macrophages. ( A ) 48 h post plasma treatment and co-culture with M0 macrophages, A549 cells showed reduced viability compared to those treated with plasma and cultured alone. ( B ) Enhanced anti-tumor effects were abrogated when ATP secretion was inhibited with DPI (Two Way ANOVA, Tukey’s multiple comparison test). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Plasma treatment of tumor cells in immunogenic cell death (ICD)-inducing regimes enhanced anti-tumor activity of macrophages. ( A ) 48 h post plasma treatment and co-culture with M0 macrophages, A549 cells showed reduced viability compared to those treated with plasma and cultured alone. ( B ) Enhanced anti-tumor effects were abrogated when ATP secretion was inhibited with DPI (Two Way ANOVA, Tukey’s multiple comparison test). Data are shown as mean ± SEM. * p

    Techniques Used: Activity Assay, Co-Culture Assay, Cell Culture

    35) Product Images from "Overexpression of amplified in breast cancer 1 (AIB1) gene promotes lung adenocarcinoma aggressiveness in vitro and in vivo by upregulating C-X-C motif chemokine receptor 4"

    Article Title: Overexpression of amplified in breast cancer 1 (AIB1) gene promotes lung adenocarcinoma aggressiveness in vitro and in vivo by upregulating C-X-C motif chemokine receptor 4

    Journal: Cancer Communications

    doi: 10.1186/s40880-018-0320-1

    Overexpression of AIB1 enhances lung adenocarcinoma A549 cell metastasis mediated by CXCR4 in vivo. a Left: representative lungs showing metastatic nodules originating from A549-vector, A549-AIB1, and A549-AIB1 + CXCR4-shRNA cells injected with Balb/c nude mice. Right: number of metastatic nodules formed in the lungs of mice 6 weeks after tail vein injection of A549-vector, A549-AIB1, and A549-AIB1 + CXCR4-shRNA cells (eight mice per group; * P
    Figure Legend Snippet: Overexpression of AIB1 enhances lung adenocarcinoma A549 cell metastasis mediated by CXCR4 in vivo. a Left: representative lungs showing metastatic nodules originating from A549-vector, A549-AIB1, and A549-AIB1 + CXCR4-shRNA cells injected with Balb/c nude mice. Right: number of metastatic nodules formed in the lungs of mice 6 weeks after tail vein injection of A549-vector, A549-AIB1, and A549-AIB1 + CXCR4-shRNA cells (eight mice per group; * P

    Techniques Used: Over Expression, In Vivo, Plasmid Preparation, shRNA, Injection, Mouse Assay

    The associations of AIB1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in lung adenocarcinoma cells. a The five genes, CXCR4, tumor necrosis factor (ligand) superfamily member 10 (TNFSF10), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 2 (MMP2), and vascular endothelial growth factor A (VEGFA), showed more than a 3.5-fold mRNA differential expression in shAIB1-transfected H1993 cells compared with that in control H1993 cells, as shown by using a human tumor metastasis RT2 profiler PCR array. b Silencing of AIB1 by two shRNAs down-regulated CXCR4 expression in shAIB1 H1993 cells, as detected by Western blotting. c Upper left: treatment of 2 CXCR4-shRNAs in A549-AIB1 cells efficiently decreased the expression levels of CXCR4 as detected by Western blotting. Upper right and down: wound-healing assay showed that the enhanced migrative ability in A549-AIB1 cells was inhibited by silencing CXCR4. d Transwell assay demonstrated that the increased invasive capacity of A549-AIB1 cells was suppressed by CXCR4 silencing. Data are the mean ± SE of three independent experiments. ** P
    Figure Legend Snippet: The associations of AIB1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in lung adenocarcinoma cells. a The five genes, CXCR4, tumor necrosis factor (ligand) superfamily member 10 (TNFSF10), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 2 (MMP2), and vascular endothelial growth factor A (VEGFA), showed more than a 3.5-fold mRNA differential expression in shAIB1-transfected H1993 cells compared with that in control H1993 cells, as shown by using a human tumor metastasis RT2 profiler PCR array. b Silencing of AIB1 by two shRNAs down-regulated CXCR4 expression in shAIB1 H1993 cells, as detected by Western blotting. c Upper left: treatment of 2 CXCR4-shRNAs in A549-AIB1 cells efficiently decreased the expression levels of CXCR4 as detected by Western blotting. Upper right and down: wound-healing assay showed that the enhanced migrative ability in A549-AIB1 cells was inhibited by silencing CXCR4. d Transwell assay demonstrated that the increased invasive capacity of A549-AIB1 cells was suppressed by CXCR4 silencing. Data are the mean ± SE of three independent experiments. ** P

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Western Blot, Wound Healing Assay, Transwell Assay

    Overexpression of AIB1 enhances lung adenocarcinoma A549 cell migration and invasion in vitro. a Left: Western blotting reveals that ectopic expression of AIB1 was substantially increased in A549-AIB1 cells compared with that in A549-vector cells (upper panel); right: representative results of wound-healing assays demonstrate that A549-AIB1 cells had higher motility than A549-vector cells. The numbers of migrating cells are shown in the left bottom panel. Data are the mean ± SE of three independent experiments; ** P
    Figure Legend Snippet: Overexpression of AIB1 enhances lung adenocarcinoma A549 cell migration and invasion in vitro. a Left: Western blotting reveals that ectopic expression of AIB1 was substantially increased in A549-AIB1 cells compared with that in A549-vector cells (upper panel); right: representative results of wound-healing assays demonstrate that A549-AIB1 cells had higher motility than A549-vector cells. The numbers of migrating cells are shown in the left bottom panel. Data are the mean ± SE of three independent experiments; ** P

    Techniques Used: Over Expression, Migration, In Vitro, Western Blot, Expressing, Plasmid Preparation

    36) Product Images from "XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway"

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8491

    MTT assays. A549 cells were treated with increasing concentration of XAV939 (0.1, 0.5, 1,5 and 10 µM) for 24, 4, 72 and 96 h. The result was shown as relative cell viability per concentration at each time point.
    Figure Legend Snippet: MTT assays. A549 cells were treated with increasing concentration of XAV939 (0.1, 0.5, 1,5 and 10 µM) for 24, 4, 72 and 96 h. The result was shown as relative cell viability per concentration at each time point.

    Techniques Used: MTT Assay, Concentration Assay

    Localization of β-catenin immunofluorescence in response to treatment with NaCl (control), 0., 1 and 10 µmol/l XAV939 in A549 cells (magnification, ×400).
    Figure Legend Snippet: Localization of β-catenin immunofluorescence in response to treatment with NaCl (control), 0., 1 and 10 µmol/l XAV939 in A549 cells (magnification, ×400).

    Techniques Used: Immunofluorescence

    c-Myc mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P
    Figure Legend Snippet: c-Myc mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Εffect of XAV939 on A549 cell migration was detected via wound-healing assay. The wound widths in the different XAV939 treatment groups (0.1, 0.5, 1, 5, 10 µmol/l) after 24 h were all significantly wider than those of the control group (magnification, ×200).
    Figure Legend Snippet: Εffect of XAV939 on A549 cell migration was detected via wound-healing assay. The wound widths in the different XAV939 treatment groups (0.1, 0.5, 1, 5, 10 µmol/l) after 24 h were all significantly wider than those of the control group (magnification, ×200).

    Techniques Used: Migration, Wound Healing Assay

    β-catenin mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P
    Figure Legend Snippet: β-catenin mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    (A) XAV939 inhibits the clonogenicity of A549 cells in vitro . (B) Treatment of the A549 cell line with 0.1, 1 and 10 µmol/l XAV939 significantly inhibited colony formation compared with the control. *P
    Figure Legend Snippet: (A) XAV939 inhibits the clonogenicity of A549 cells in vitro . (B) Treatment of the A549 cell line with 0.1, 1 and 10 µmol/l XAV939 significantly inhibited colony formation compared with the control. *P

    Techniques Used: In Vitro

    Expression of TNKS in three lung adenocarcinoma cell lines. (A) Western blot analysis and (B) quantification of this analysis demonstrated that the level of TNKS protein expression in A549 cells was significantly higher compared with that in Calu-3 and SK-LU-1 cells. *P
    Figure Legend Snippet: Expression of TNKS in three lung adenocarcinoma cell lines. (A) Western blot analysis and (B) quantification of this analysis demonstrated that the level of TNKS protein expression in A549 cells was significantly higher compared with that in Calu-3 and SK-LU-1 cells. *P

    Techniques Used: Expressing, Western Blot

    37) Product Images from "miR-577 suppresses cell proliferation and epithelial-mesenchymal transition by regulating the WNT2B mediated Wnt/β-catenin pathway in non-small cell lung cancer"

    Article Title: miR-577 suppresses cell proliferation and epithelial-mesenchymal transition by regulating the WNT2B mediated Wnt/β-catenin pathway in non-small cell lung cancer

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.9279

    miR-577 negatively regulates the Wnt/β-catenin pathway. (A) Immunofluorescence staining was used to examine the nuclear distribution of β-catenin in A549 cells. Original magnification, ×100. (B) Western blot analysis was conducted to detect the expression levels of p-GSK3β, total-GSK3β, β-catenin, c-Myc and cyclin D1 in A549 cells. (C) TOP/FOP luciferase reporter assays were performed to detect the β-catenin activity in H522 and A549 cells. (D) Regulation model of miR-577. *P
    Figure Legend Snippet: miR-577 negatively regulates the Wnt/β-catenin pathway. (A) Immunofluorescence staining was used to examine the nuclear distribution of β-catenin in A549 cells. Original magnification, ×100. (B) Western blot analysis was conducted to detect the expression levels of p-GSK3β, total-GSK3β, β-catenin, c-Myc and cyclin D1 in A549 cells. (C) TOP/FOP luciferase reporter assays were performed to detect the β-catenin activity in H522 and A549 cells. (D) Regulation model of miR-577. *P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Expressing, Luciferase, Activity Assay

    WNT2B promotes the malignant phenotype of non-small cell lung cancer cells. (A) mRNA expression levels of WNT2B in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by RT-qPCR. (B) Protein expression levels of WNT2B in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by western blot analysis. (C) RT-qPCR demonstrated that overexpression and knockdown of WNT2B was efficient. (D) Western blot analysis revealed that overexpression and knockdown of WNT2B was efficient in A549 cells. (E) CCK-8 assay demonstrated that WNT2B overexpression increased cell viability and WNT2B knockdown decreased cell viability. (F) Relative colony formation rate was higher following transfection with pWNT2B and lower with shR-WNT2B compared with the control groups. Original magnification, ×1. (G) Transwell migration assays revealed that WNT2B overexpression promoted cell migration ability. Original magnification, ×10. (H) Transwell invasion assays revealed that WNT2B overexpression promoted cell invasion ability. Original magnification, ×10. (I) Western blot analysis demonstrated the protein expression levels of E-cadherin, ICAM-1 and Vimentin, following transfection with pWNT2B or shR-WNT2B in A549 cells. *P
    Figure Legend Snippet: WNT2B promotes the malignant phenotype of non-small cell lung cancer cells. (A) mRNA expression levels of WNT2B in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by RT-qPCR. (B) Protein expression levels of WNT2B in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by western blot analysis. (C) RT-qPCR demonstrated that overexpression and knockdown of WNT2B was efficient. (D) Western blot analysis revealed that overexpression and knockdown of WNT2B was efficient in A549 cells. (E) CCK-8 assay demonstrated that WNT2B overexpression increased cell viability and WNT2B knockdown decreased cell viability. (F) Relative colony formation rate was higher following transfection with pWNT2B and lower with shR-WNT2B compared with the control groups. Original magnification, ×1. (G) Transwell migration assays revealed that WNT2B overexpression promoted cell migration ability. Original magnification, ×10. (H) Transwell invasion assays revealed that WNT2B overexpression promoted cell invasion ability. Original magnification, ×10. (I) Western blot analysis demonstrated the protein expression levels of E-cadherin, ICAM-1 and Vimentin, following transfection with pWNT2B or shR-WNT2B in A549 cells. *P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, CCK-8 Assay, Transfection, Migration

    miR-577 functions as a suppressor gene in NSCLC cells. (A) Expression levels of miR-577 in NSCLC and normal tissues were examined by RT-qPCR. (B) Expression levels of miR-577 in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by RT-qPCR. (C) Efficiency of pri-miR-577 or ASO-miR-577 was identified by RT-qPCR. (D) Effect of miR-577 on H522 and A549 cellular viabilities were determined by CCK-8 assay. (E) Relative colony formation rates of H522 and A549 cells with indicated treatment were determined by colony formation assay. Original magnification, ×1. (F) Transwell migration assays revealed that miR-577 suppressed cell migration ability. Original magnification, ×10. (G) Transwell invasion assays demonstrated that miR-577 suppressed cell invasion ability. Original magnification, ×10. (H) Western blot analysis of the protein exp ression levels of E-cadherin, ICAM-1 and Vimentin following transfection with pri-miR-577 or ASO-miR-577 and the control groups in A549 cells. *P
    Figure Legend Snippet: miR-577 functions as a suppressor gene in NSCLC cells. (A) Expression levels of miR-577 in NSCLC and normal tissues were examined by RT-qPCR. (B) Expression levels of miR-577 in HBECs, H650, A549, H522, H1299 and H1155 cells were examined by RT-qPCR. (C) Efficiency of pri-miR-577 or ASO-miR-577 was identified by RT-qPCR. (D) Effect of miR-577 on H522 and A549 cellular viabilities were determined by CCK-8 assay. (E) Relative colony formation rates of H522 and A549 cells with indicated treatment were determined by colony formation assay. Original magnification, ×1. (F) Transwell migration assays revealed that miR-577 suppressed cell migration ability. Original magnification, ×10. (G) Transwell invasion assays demonstrated that miR-577 suppressed cell invasion ability. Original magnification, ×10. (H) Western blot analysis of the protein exp ression levels of E-cadherin, ICAM-1 and Vimentin following transfection with pri-miR-577 or ASO-miR-577 and the control groups in A549 cells. *P

    Techniques Used: Expressing, Quantitative RT-PCR, Allele-specific Oligonucleotide, CCK-8 Assay, Colony Assay, Migration, Western Blot, Transfection

    WNT2B is a direct target of miR-577. (A) Predicted miR-577 binding sites in WNT2B mRNA using TargetScan were demonstrated and the mutated 3′UTR of WNT2B mRNA is shown. EGFP intensity of H522 and A549 cells expression the (B) wild type or (C) mutated 3′UTR was determined by spectrophotometry, and the values of control group were set to 1. (D) WNT2B mRNA expression levels in H522 and A549 cells with indicated treatment were measured by RT-qPCR. (E) WNT2B protein level in H522 and A549 cells transfected with pri-miR-577 or ASO-miR-577 and respective controls were determined by western blot analysis. (F) mRNA expression levels of WNT2B was negatively correlated to the miR-577 level by RT-qPCR. *P
    Figure Legend Snippet: WNT2B is a direct target of miR-577. (A) Predicted miR-577 binding sites in WNT2B mRNA using TargetScan were demonstrated and the mutated 3′UTR of WNT2B mRNA is shown. EGFP intensity of H522 and A549 cells expression the (B) wild type or (C) mutated 3′UTR was determined by spectrophotometry, and the values of control group were set to 1. (D) WNT2B mRNA expression levels in H522 and A549 cells with indicated treatment were measured by RT-qPCR. (E) WNT2B protein level in H522 and A549 cells transfected with pri-miR-577 or ASO-miR-577 and respective controls were determined by western blot analysis. (F) mRNA expression levels of WNT2B was negatively correlated to the miR-577 level by RT-qPCR. *P

    Techniques Used: Binding Assay, Expressing, Spectrophotometry, Quantitative RT-PCR, Transfection, Allele-specific Oligonucleotide, Western Blot

    38) Product Images from "Theabrownin Inhibits Cell Cycle Progression and Tumor Growth of Lung Carcinoma through c-myc-Related Mechanism"

    Article Title: Theabrownin Inhibits Cell Cycle Progression and Tumor Growth of Lung Carcinoma through c-myc-Related Mechanism

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00075

    Relative mRNA expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h. Values are presented as mean ± SD of three replicates. Different symbols (a, ab, b, c, bc, d) indicate significant difference between groups [Fisher’s least significant difference (LSD), P
    Figure Legend Snippet: Relative mRNA expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h. Values are presented as mean ± SD of three replicates. Different symbols (a, ab, b, c, bc, d) indicate significant difference between groups [Fisher’s least significant difference (LSD), P

    Techniques Used: Expressing

    c-myc-related mechanism of TB on A549 cells .
    Figure Legend Snippet: c-myc-related mechanism of TB on A549 cells .

    Techniques Used:

    Effect of TB on cell cycle of A549 cells determined by flow cytometry. Values were presented as mean ± SD ( n = 3). ∗ P
    Figure Legend Snippet: Effect of TB on cell cycle of A549 cells determined by flow cytometry. Values were presented as mean ± SD ( n = 3). ∗ P

    Techniques Used: Flow Cytometry, Cytometry

    Protein expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h .
    Figure Legend Snippet: Protein expression of target genes in A549 cells with TB treatment at 200 μg/ml for 24, 48, and 72 h .

    Techniques Used: Expressing

    Effect of Theabrownin (TB) and green tea extract on cell viability of A549 cells determined by MTT assay. Values were presented as mean ± SD ( n = 5).
    Figure Legend Snippet: Effect of Theabrownin (TB) and green tea extract on cell viability of A549 cells determined by MTT assay. Values were presented as mean ± SD ( n = 5).

    Techniques Used: MTT Assay

    39) Product Images from "Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells"

    Article Title: Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells

    Journal:

    doi: 10.1152/ajplung.00052.2009

    Expression of green fluorescence protein (GFP)-tagged Prdx6 in lysosome-related compartments of MLE-12 and A549 lung epithelial cells. Left panels : GFP full-length Prdx6 fusion construct (FL) and GFP control (vector) expressed in transfected MLE-12 (
    Figure Legend Snippet: Expression of green fluorescence protein (GFP)-tagged Prdx6 in lysosome-related compartments of MLE-12 and A549 lung epithelial cells. Left panels : GFP full-length Prdx6 fusion construct (FL) and GFP control (vector) expressed in transfected MLE-12 (

    Techniques Used: Expressing, Fluorescence, Construct, Plasmid Preparation, Transfection

    Expression of GFP-tagged Prdx6 deletion mutants in MLE-12 and A549 lung epithelial cells. A : schematic representation of GFP:Prdx6 deletion constructs. B : colocalization of each of the GFP:Prdx6 deletion mutants with Nile Red in MLE-12 cells. C : colocalization
    Figure Legend Snippet: Expression of GFP-tagged Prdx6 deletion mutants in MLE-12 and A549 lung epithelial cells. A : schematic representation of GFP:Prdx6 deletion constructs. B : colocalization of each of the GFP:Prdx6 deletion mutants with Nile Red in MLE-12 cells. C : colocalization

    Techniques Used: Expressing, Construct

    40) Product Images from "NORAD accelerates chemo-resistance of non-small-cell lung cancer via targeting at miR-129-1-3p/SOX4 axis"

    Article Title: NORAD accelerates chemo-resistance of non-small-cell lung cancer via targeting at miR-129-1-3p/SOX4 axis

    Journal: Bioscience Reports

    doi: 10.1042/BSR20193489

    NORAD knockdown enhanced DDP sensitivity of H446/DDP and A549/DPP cells via restricting SOX4 expression ( A ) SOX4 expression was tested in cells transfected with pcDNA3.1/SOX4. ( B and C ) MMT and colony formation assays were conducted to measure cell proliferation in cells transfected with sh-NORAD#1 and pcDNA3.1/SOX4 treated with DDP MMT assay revealed the IC50 value for cells under indicted transfections( D and E ) Flow cytometry analysis and TUNEL were carried out to measure apoptosis rate in cells transfected with sh-NORAD#1 and pcDNA3.1/SOX4 treated with DDP; ** P
    Figure Legend Snippet: NORAD knockdown enhanced DDP sensitivity of H446/DDP and A549/DPP cells via restricting SOX4 expression ( A ) SOX4 expression was tested in cells transfected with pcDNA3.1/SOX4. ( B and C ) MMT and colony formation assays were conducted to measure cell proliferation in cells transfected with sh-NORAD#1 and pcDNA3.1/SOX4 treated with DDP MMT assay revealed the IC50 value for cells under indicted transfections( D and E ) Flow cytometry analysis and TUNEL were carried out to measure apoptosis rate in cells transfected with sh-NORAD#1 and pcDNA3.1/SOX4 treated with DDP; ** P

    Techniques Used: Expressing, Transfection, Flow Cytometry, TUNEL Assay

    NORAD expression was lifted in NSCLC tissues and cells ( A ) RT-qPCR assays were used to test the expression of NORAD in NSCLC tissues and adjacent normal tissues. ( B ) NORAD expression was examined in DDP-sensitive and DDP-resistant tissues. ( C ) Kaplan–Meier curve was used to analyze the overall survival between high and low NORAD expression. ( D ) N0RAD expression was tested in NSCLC cell lines (H446 and A549) and normal human lung bronchial epithelial BEAS-2B cell as well as their DDP-resistant cell H446/DDP and A549/DDP cells. ( E ) MMT was conducted to evaluate cell viability of H446 and A549 while their parental cells were treated with DDP. ( F ) RT-qPCR assays were conducted to appraise the efficiency of sh-NORAD#1/2 in cells. ( G and H ) MTT and colony formation assays were conducted to examine the cell proliferation while H446/DDP and A549/DDP transfected with sh-NORAD#1/2 exposed to DDP. ( I and J ) Flow cytometry analysis and TUNEL were conducted to probe rate of apoptosis in H446/DDP and A549/DDP transfected with sh-NORAD#1/2 after exposure of 2 μg/ml DDP; ** P
    Figure Legend Snippet: NORAD expression was lifted in NSCLC tissues and cells ( A ) RT-qPCR assays were used to test the expression of NORAD in NSCLC tissues and adjacent normal tissues. ( B ) NORAD expression was examined in DDP-sensitive and DDP-resistant tissues. ( C ) Kaplan–Meier curve was used to analyze the overall survival between high and low NORAD expression. ( D ) N0RAD expression was tested in NSCLC cell lines (H446 and A549) and normal human lung bronchial epithelial BEAS-2B cell as well as their DDP-resistant cell H446/DDP and A549/DDP cells. ( E ) MMT was conducted to evaluate cell viability of H446 and A549 while their parental cells were treated with DDP. ( F ) RT-qPCR assays were conducted to appraise the efficiency of sh-NORAD#1/2 in cells. ( G and H ) MTT and colony formation assays were conducted to examine the cell proliferation while H446/DDP and A549/DDP transfected with sh-NORAD#1/2 exposed to DDP. ( I and J ) Flow cytometry analysis and TUNEL were conducted to probe rate of apoptosis in H446/DDP and A549/DDP transfected with sh-NORAD#1/2 after exposure of 2 μg/ml DDP; ** P

    Techniques Used: Expressing, Quantitative RT-PCR, MTT Assay, Transfection, Flow Cytometry, TUNEL Assay

    MiR-129-1-3p overexpression weakened DDP resistance in NSCLC ( A and B ) MTT revealed the IC50 value in DDP-resistant cells transfected with miR-129-1-3p mimics; and colony formation assays were used to test cell proliferation and H446/DDP and A549/DDP cells treated with miR-129-1-3p mimics;. ( C and D ) Flow cytometry analysis and TUNEL were applied to examine apoptosis in cells transfected with miR-129-1-3p mimics and treated with DDP. ( E ) MiR-129-1-3p expression was detected in cells transfected with miR-129-1-3p inhibitor. ( F and G ) MTT and colony formation assays were carried out to determine cell proliferation in cells transfected with sh-NORAD#1 and miR-129-1-3p inhibitor and treated with DDP. ( H and I ) Flow cytometry analysis and TUNEL were carried out to examine apoptosis rate in cells transfected with sh-NORAD#1 and miR-129-1-3p inhibitor and treated with DDP; ** P
    Figure Legend Snippet: MiR-129-1-3p overexpression weakened DDP resistance in NSCLC ( A and B ) MTT revealed the IC50 value in DDP-resistant cells transfected with miR-129-1-3p mimics; and colony formation assays were used to test cell proliferation and H446/DDP and A549/DDP cells treated with miR-129-1-3p mimics;. ( C and D ) Flow cytometry analysis and TUNEL were applied to examine apoptosis in cells transfected with miR-129-1-3p mimics and treated with DDP. ( E ) MiR-129-1-3p expression was detected in cells transfected with miR-129-1-3p inhibitor. ( F and G ) MTT and colony formation assays were carried out to determine cell proliferation in cells transfected with sh-NORAD#1 and miR-129-1-3p inhibitor and treated with DDP. ( H and I ) Flow cytometry analysis and TUNEL were carried out to examine apoptosis rate in cells transfected with sh-NORAD#1 and miR-129-1-3p inhibitor and treated with DDP; ** P

    Techniques Used: Over Expression, MTT Assay, Transfection, Flow Cytometry, TUNEL Assay, Expressing

    SOX4 worked as a functional target of miR-129-1-3p in NSCLC ( A ) The candidate miRNAs expressions were detected by RT-qPCR assays in cells transfected with miR-129-1-3p mimics. ( B ) Bioinformatics predicted putative binding sites between miR-1291-1-3p and SOX4. ( C ) SOX4 expression and protein were detected in cells transfected with miR-129-1-3p mimics. ( D ) RIP assays were conducted to confirm miR-129-1-3p and SOX4 coexisted in RISCs. ( E ) The overexpressing efficiency of pcDNA3.1/NORAD was evaluated by RT-qPCR assay. ( F ) RNA pull-down assays were conducted to investigate the effects of NORAD overexpression on the interaction between miR-129-1-3p and SOX4 in H446/DDP and A549/DDP cells. ( G ) Luciferase reporter assays were conducted to verify the combination between miR-129-1-3p and SOX4; * P
    Figure Legend Snippet: SOX4 worked as a functional target of miR-129-1-3p in NSCLC ( A ) The candidate miRNAs expressions were detected by RT-qPCR assays in cells transfected with miR-129-1-3p mimics. ( B ) Bioinformatics predicted putative binding sites between miR-1291-1-3p and SOX4. ( C ) SOX4 expression and protein were detected in cells transfected with miR-129-1-3p mimics. ( D ) RIP assays were conducted to confirm miR-129-1-3p and SOX4 coexisted in RISCs. ( E ) The overexpressing efficiency of pcDNA3.1/NORAD was evaluated by RT-qPCR assay. ( F ) RNA pull-down assays were conducted to investigate the effects of NORAD overexpression on the interaction between miR-129-1-3p and SOX4 in H446/DDP and A549/DDP cells. ( G ) Luciferase reporter assays were conducted to verify the combination between miR-129-1-3p and SOX4; * P

    Techniques Used: Functional Assay, Quantitative RT-PCR, Transfection, Binding Assay, Expressing, Over Expression, Luciferase

    Related Articles

    Transfection:

    Article Title: PAK1 Kinase Promotes Cell Motility and Invasiveness through CRK-II Serine Phosphorylation in Non-Small Cell Lung Cancer Cells
    Article Snippet: .. Subsequently, A549 cells were transfected with the above mentioned plasmids by using Lipofectamine 2000 (Invitrogen). .. 24 hours after transfection, cells were treated with G418 at 1500 µg/ml in order to generate stable cell lines.

    Article Title: Requirement of a Novel Splicing Variant of Human Histone Deacetylase 6 for TGF-?1-mediated Gene Activation
    Article Snippet: .. A549 cells were transfected with the HDAC6 expression vectors, or the reporter constructs (3TP-LUX and RL-TK), and/or siRNAs using Lipofectamine 2000 (Invitrogen) as previously described [ ]. .. In the selected transfections, A549 cells were exposed to TGF-β1 as described above.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells
    Article Snippet: .. RT-PCR Total RNA was isolated from the A549 cells with Trizol reagent (Invitrogen, Grand Island, NY, USA). .. PCR was performed with a DNA thermal cycler in a 50-μL reaction volume, containing 5 μL 10× Taq Buffer, 4 μL of 2.5 mM dNTP, 4 μL of 25 mM MgCl2 , 2 μL each forward and backward primers, 0.5 μL Taq polymerase, and 2 μL cDNA template, for 35 cycles by using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    Stable Transfection:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Synthesized:

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells
    Article Snippet: .. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from BEAS-2B, H1299 and A549 cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from total RNA (1,000 ng) using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the temperature protocol of 25°C for 10 min, 37°C for 120 min and 85°C for 5min. qPCR was performed using the SYBR Master Mix (Roche Diagnostics, Basel, Switzerland). ..

    Isolation:

    Article Title: Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1? under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells
    Article Snippet: .. RT-PCR Total RNA was isolated from the A549 cells with Trizol reagent (Invitrogen, Grand Island, NY, USA). .. PCR was performed with a DNA thermal cycler in a 50-μL reaction volume, containing 5 μL 10× Taq Buffer, 4 μL of 2.5 mM dNTP, 4 μL of 25 mM MgCl2 , 2 μL each forward and backward primers, 0.5 μL Taq polymerase, and 2 μL cDNA template, for 35 cycles by using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells
    Article Snippet: .. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from BEAS-2B, H1299 and A549 cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from total RNA (1,000 ng) using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the temperature protocol of 25°C for 10 min, 37°C for 120 min and 85°C for 5min. qPCR was performed using the SYBR Master Mix (Roche Diagnostics, Basel, Switzerland). ..

    Construct:

    Article Title: Requirement of a Novel Splicing Variant of Human Histone Deacetylase 6 for TGF-?1-mediated Gene Activation
    Article Snippet: .. A549 cells were transfected with the HDAC6 expression vectors, or the reporter constructs (3TP-LUX and RL-TK), and/or siRNAs using Lipofectamine 2000 (Invitrogen) as previously described [ ]. .. In the selected transfections, A549 cells were exposed to TGF-β1 as described above.

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Real-time Polymerase Chain Reaction:

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells
    Article Snippet: .. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from BEAS-2B, H1299 and A549 cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from total RNA (1,000 ng) using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the temperature protocol of 25°C for 10 min, 37°C for 120 min and 85°C for 5min. qPCR was performed using the SYBR Master Mix (Roche Diagnostics, Basel, Switzerland). ..

    Concentration Assay:

    Article Title: Health hazards of methylammonium lead iodide based perovskites: cytotoxicity studies hazards of methylammonium lead iodide based perovskites: cytotoxicity studies †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tx00303b
    Article Snippet: .. A549 cells were plated in 24-well plates and the medium was replaced by PBS containing CFSE proliferation dye (Life Technologies) at a final concentration of 5 μM and left for 10 minutes at 37 °C then rinsed with PBS and left in complete medium overnight. ..

    Incubation:

    Article Title: Inhibition of CK2? Down-Regulates Hedgehog/Gli Signaling Leading to a Reduction of a Stem-Like Side Population in Human Lung Cancer Cells
    Article Snippet: .. The A549 cells were incubated with 5 µg/ml Hoechst 33342 dye (Invitrogen) for 90 min with and without 50 µM verapamil. .. Cell samples were analyzed and sorted using a Moflo MLS cell sorter (Beckman-Coulter, Hialeah, FL) with UV capabilities and SUMMIT software for data acquisition and analysis.

    Quantitative RT-PCR:

    Article Title: Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6
    Article Snippet: .. Quantitative RT-PCR HeLa and A549 cells grown in 10-cm dishes were washed with phosphate-buffered sa line before their total RNA were extracted with TRIzol reagent (Life Technologies) as per the manufacturer’s instructions. .. The total RNA were reverse transcribed with random hexamers and Superscript III MMLV RT (Life Technologies) according to the manufacturer’s instructions.

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells
    Article Snippet: .. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from BEAS-2B, H1299 and A549 cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from total RNA (1,000 ng) using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the temperature protocol of 25°C for 10 min, 37°C for 120 min and 85°C for 5min. qPCR was performed using the SYBR Master Mix (Roche Diagnostics, Basel, Switzerland). ..

    Expressing:

    Article Title: Requirement of a Novel Splicing Variant of Human Histone Deacetylase 6 for TGF-?1-mediated Gene Activation
    Article Snippet: .. A549 cells were transfected with the HDAC6 expression vectors, or the reporter constructs (3TP-LUX and RL-TK), and/or siRNAs using Lipofectamine 2000 (Invitrogen) as previously described [ ]. .. In the selected transfections, A549 cells were exposed to TGF-β1 as described above.

    Polymerase Chain Reaction:

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells
    Article Snippet: .. RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from BEAS-2B, H1299 and A549 cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from total RNA (1,000 ng) using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the temperature protocol of 25°C for 10 min, 37°C for 120 min and 85°C for 5min. qPCR was performed using the SYBR Master Mix (Roche Diagnostics, Basel, Switzerland). ..

    Plasmid Preparation:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

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    Thermo Fisher human lung cancer a549
    Tetracenomycin X decreases the expression of cyclin D1 by the activation of p38 and c-JUN. ( A ) The <t>A549</t> and H460 cells were treated with tetracenomycin X for the indicated times or treated with various concentrations of tetracenomycin X for 8 h. The expression levels of the proteins were determined using western blotting. ( B ) The viability of the A5459 and H460 cells was determined after a 4-h pre-treatment with SP600125 or SB203580 and an 8-h treatment with tetracenomycin X. The expression levels of the proteins were determined using western blotting.
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    Thermo Fisher a549
    Identification of host factors that promote the replication of chimeric yellow fever (YFV)/ West Nile (WNV) reporter particles. ( A ) A panel of mammalian cell lines was infected with YFV at a multiplicity of infection (MOI) of 1 for 24 h. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis. ( B ) <t>A549</t> and HT1080 cells were infected with WNV or Zika (ZIKV) at an MOI of 1. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis 24 h post-infection. ( C ) Schematic depiction of YFV/WNV chimeric reporter viral particles (RVPs). ( D ) Schematic depiction of the genome-wide gain-of-function cDNA screening approach. ( E ) Candidate genes identified by next-generation sequencing of pooled-colony genomic DNA. The Y-axis represents the number of reads that map to each gene, represented by a circle. ( F ) The genes with more than ten reads were classified according to subcellular localization. The Y-axis represents the number of reads that map to each gene. Data in ( A ) and ( B ) are represented as mean ± SD of at least three independent experiments. The red line in ( E ) and ( F ) marks a cut-off value of 100 reads per mapped gene.
    A549, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher human a549 cells
    FUS/TLS accumulation at sites of UVA laser-induced damage is dependent on PAR synthesis. ( A ) Human <t>A549</t> cells were transfected with GFP-hFUS and microirradiated with UVA. Cells were pretreated with vehicle (DMSO) or 500 nM KU58948 1 h before microirradiation. A representative experiment is shown with quantification of GFP-FUS recruitment (mean ± SEM > 30 cells) plotted graphically (bottom left). ( B ) U2OS cells mock-treated or pretreated with 1 µM KU58948 (PARPi) were microirradiated as described above, fixed and immunolabelled for endogenous FUS (middle row) and γH2Ax (bottom).
    Human A549 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher a549 920
    The synergistic cytotoxic effect of the sequential combination therapy was correlated with the p53 status of the cell A. Combination index for each CDDP concentration after sequential combination therapy in the p53 wild type cell lines A549, A549-NTC, the p53 deficient cell line <t>A549-920</t> and the p53 mutant cell line CRL-5908. The supporting data for this figure (Mean IC 50 -values and mean CI) can be found in table 2 . B. Protein expression levels of p53 and its main transcription targets MDM2, p21, PUMA, and BAX after monotherapy with CDDP or 5 μM Nutlin-3 or sequential combination therapy in each cell line. C. Percentage of Annexin V PerCP positive cells after treatment in all cell lines, measured by flowcytometric analysis D. Cell cycles distribution after treatment as previously described in all cell lines. Cells were stained with PI and DNA content was measured by flowcytometric analysis. Cells were divided in 3 groups: G1 phase (2n); S-phase (2n-4n); and G2/M phase (4n). (* p
    A549 920, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tetracenomycin X decreases the expression of cyclin D1 by the activation of p38 and c-JUN. ( A ) The A549 and H460 cells were treated with tetracenomycin X for the indicated times or treated with various concentrations of tetracenomycin X for 8 h. The expression levels of the proteins were determined using western blotting. ( B ) The viability of the A5459 and H460 cells was determined after a 4-h pre-treatment with SP600125 or SB203580 and an 8-h treatment with tetracenomycin X. The expression levels of the proteins were determined using western blotting.

    Journal: Marine Drugs

    Article Title: Tetracenomycin X Exerts Antitumour Activity in Lung Cancer Cells through the Downregulation of Cyclin D1

    doi: 10.3390/md17010063

    Figure Lengend Snippet: Tetracenomycin X decreases the expression of cyclin D1 by the activation of p38 and c-JUN. ( A ) The A549 and H460 cells were treated with tetracenomycin X for the indicated times or treated with various concentrations of tetracenomycin X for 8 h. The expression levels of the proteins were determined using western blotting. ( B ) The viability of the A5459 and H460 cells was determined after a 4-h pre-treatment with SP600125 or SB203580 and an 8-h treatment with tetracenomycin X. The expression levels of the proteins were determined using western blotting.

    Article Snippet: Cell Culture The human lung cancer A549, H460, H157, HCC827 and H1975 cell lines were kept in our laboratory and maintained in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL) (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

    Techniques: Expressing, Activation Assay, Western Blot

    The antitumour activity of tetracenomycin X is independent of apoptosis and autophagy. ( A , C ) The A549 and H460 cells were treated with tetracenomycin X (2.5 and 5 µmol/L) for 8 h. The expression levels of the proteins were determined using western blotting. ( B ) The A549 and H460 cells were treated with tetracenomycin X (10 µmol/L) for 24 h. Apoptosis was detected by annexin V-FITC/PI staining and flow cytometry analysis.

    Journal: Marine Drugs

    Article Title: Tetracenomycin X Exerts Antitumour Activity in Lung Cancer Cells through the Downregulation of Cyclin D1

    doi: 10.3390/md17010063

    Figure Lengend Snippet: The antitumour activity of tetracenomycin X is independent of apoptosis and autophagy. ( A , C ) The A549 and H460 cells were treated with tetracenomycin X (2.5 and 5 µmol/L) for 8 h. The expression levels of the proteins were determined using western blotting. ( B ) The A549 and H460 cells were treated with tetracenomycin X (10 µmol/L) for 24 h. Apoptosis was detected by annexin V-FITC/PI staining and flow cytometry analysis.

    Article Snippet: Cell Culture The human lung cancer A549, H460, H157, HCC827 and H1975 cell lines were kept in our laboratory and maintained in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL) (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

    Techniques: Activity Assay, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry

    Tetracenomycin X induces the proteasomal degradation of cyclin D1. ( A ) RT-PCR analysis of the gene expression of cyclin D1 in the A549 cells and H460 cells. ( B ) The A549 cells and H460 cells were pre-treated with MG132 at the indicated concentration for 2 h and then co-treated with tetracenomycin X (5 µmol/L) for 12 h. The A549 cells and H460 cells were pre-treated with DMSO or tetracenomycin X (5 µmol/L) and then co-treated with 10 μg/mL of cycloheximide (CHX) for the indicated times.

    Journal: Marine Drugs

    Article Title: Tetracenomycin X Exerts Antitumour Activity in Lung Cancer Cells through the Downregulation of Cyclin D1

    doi: 10.3390/md17010063

    Figure Lengend Snippet: Tetracenomycin X induces the proteasomal degradation of cyclin D1. ( A ) RT-PCR analysis of the gene expression of cyclin D1 in the A549 cells and H460 cells. ( B ) The A549 cells and H460 cells were pre-treated with MG132 at the indicated concentration for 2 h and then co-treated with tetracenomycin X (5 µmol/L) for 12 h. The A549 cells and H460 cells were pre-treated with DMSO or tetracenomycin X (5 µmol/L) and then co-treated with 10 μg/mL of cycloheximide (CHX) for the indicated times.

    Article Snippet: Cell Culture The human lung cancer A549, H460, H157, HCC827 and H1975 cell lines were kept in our laboratory and maintained in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL) (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay

    Tetracenomycin X-induced cell cycle arrest in the G0/G1 phase and decreased the expression levels of cell cycle-related proteins in the lung cancer cells. ( A ) the A549 and H460 cells were treated with tetracenomycin X (5 or 10 µmol/L) for 16 h. The cell cycle was detected by flow cytometry analysis. ( B ) The A549 and H460 cells were treated with tetracenomycin X for the indicated times or treated with various concentrations of tetracenomycin X for 8 h. The expression levels of proteins were determined using western blotting.

    Journal: Marine Drugs

    Article Title: Tetracenomycin X Exerts Antitumour Activity in Lung Cancer Cells through the Downregulation of Cyclin D1

    doi: 10.3390/md17010063

    Figure Lengend Snippet: Tetracenomycin X-induced cell cycle arrest in the G0/G1 phase and decreased the expression levels of cell cycle-related proteins in the lung cancer cells. ( A ) the A549 and H460 cells were treated with tetracenomycin X (5 or 10 µmol/L) for 16 h. The cell cycle was detected by flow cytometry analysis. ( B ) The A549 and H460 cells were treated with tetracenomycin X for the indicated times or treated with various concentrations of tetracenomycin X for 8 h. The expression levels of proteins were determined using western blotting.

    Article Snippet: Cell Culture The human lung cancer A549, H460, H157, HCC827 and H1975 cell lines were kept in our laboratory and maintained in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL) (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

    Techniques: Expressing, Flow Cytometry, Cytometry, Western Blot

    Tetracenomycin X selectively inhibits the cell proliferation of lung cancer cells. ( A ) The structure of tetracenomycin X. ( B ) The proliferative activity of the five lung cancer cells and lung fibroblasts after being treated with tetracenomycin X and Adriamycin (0.1563, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 15 µmol/L) for 24 h was assessed by sulforhodamine B (SRB) assay. The survival rates were calculated as a ratio compared with the control group (untreated cells). The values represent the mean ± SD of three independent samples. Each experiment was repeated three times under each condition. ( C ) The cell morphology of the A549 and H460 cells under a 4 *0.1 microscope after treatment with tetracenomycin X for 24 h.

    Journal: Marine Drugs

    Article Title: Tetracenomycin X Exerts Antitumour Activity in Lung Cancer Cells through the Downregulation of Cyclin D1

    doi: 10.3390/md17010063

    Figure Lengend Snippet: Tetracenomycin X selectively inhibits the cell proliferation of lung cancer cells. ( A ) The structure of tetracenomycin X. ( B ) The proliferative activity of the five lung cancer cells and lung fibroblasts after being treated with tetracenomycin X and Adriamycin (0.1563, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 15 µmol/L) for 24 h was assessed by sulforhodamine B (SRB) assay. The survival rates were calculated as a ratio compared with the control group (untreated cells). The values represent the mean ± SD of three independent samples. Each experiment was repeated three times under each condition. ( C ) The cell morphology of the A549 and H460 cells under a 4 *0.1 microscope after treatment with tetracenomycin X for 24 h.

    Article Snippet: Cell Culture The human lung cancer A549, H460, H157, HCC827 and H1975 cell lines were kept in our laboratory and maintained in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 µg/mL) (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

    Techniques: Activity Assay, Sulforhodamine B Assay, Microscopy

    Identification of host factors that promote the replication of chimeric yellow fever (YFV)/ West Nile (WNV) reporter particles. ( A ) A panel of mammalian cell lines was infected with YFV at a multiplicity of infection (MOI) of 1 for 24 h. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis. ( B ) A549 and HT1080 cells were infected with WNV or Zika (ZIKV) at an MOI of 1. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis 24 h post-infection. ( C ) Schematic depiction of YFV/WNV chimeric reporter viral particles (RVPs). ( D ) Schematic depiction of the genome-wide gain-of-function cDNA screening approach. ( E ) Candidate genes identified by next-generation sequencing of pooled-colony genomic DNA. The Y-axis represents the number of reads that map to each gene, represented by a circle. ( F ) The genes with more than ten reads were classified according to subcellular localization. The Y-axis represents the number of reads that map to each gene. Data in ( A ) and ( B ) are represented as mean ± SD of at least three independent experiments. The red line in ( E ) and ( F ) marks a cut-off value of 100 reads per mapped gene.

    Journal: Viruses

    Article Title: Uncovering Flavivirus Host Dependency Factors through a Genome-Wide Gain-of-Function Screen

    doi: 10.3390/v11010068

    Figure Lengend Snippet: Identification of host factors that promote the replication of chimeric yellow fever (YFV)/ West Nile (WNV) reporter particles. ( A ) A panel of mammalian cell lines was infected with YFV at a multiplicity of infection (MOI) of 1 for 24 h. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis. ( B ) A549 and HT1080 cells were infected with WNV or Zika (ZIKV) at an MOI of 1. The percentages of cells that expressed the viral E protein were determined by flow cytometry analysis 24 h post-infection. ( C ) Schematic depiction of YFV/WNV chimeric reporter viral particles (RVPs). ( D ) Schematic depiction of the genome-wide gain-of-function cDNA screening approach. ( E ) Candidate genes identified by next-generation sequencing of pooled-colony genomic DNA. The Y-axis represents the number of reads that map to each gene, represented by a circle. ( F ) The genes with more than ten reads were classified according to subcellular localization. The Y-axis represents the number of reads that map to each gene. Data in ( A ) and ( B ) are represented as mean ± SD of at least three independent experiments. The red line in ( E ) and ( F ) marks a cut-off value of 100 reads per mapped gene.

    Article Snippet: A549, HeLa, HEK293T and BHK-21 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Thermo Fischer Scientific, Waltham, MA, USA).

    Techniques: Infection, Flow Cytometry, Cytometry, Genome Wide, Next-Generation Sequencing

    Ribosomal protein L19 (RPL19) is required for YFV and WNV viral protein synthesis. ( A ) HeLa cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected 48 h later with YFV, WNV, or ZIKV at an MOI of 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells and normalized to cells transfected with non-targeting control siRNA. ( B ) HeLa cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected with YFV, WNV, or ZIKV 48 h later at an MOI of 1. The relative amounts of cell-associated viral RNA were determined by qPCR analysis at 24 h post-infection. Amounts of viral RNA are expressed as genome equivalents (GE) per μg of total cellular RNA and normalized to the sample transfected with non-targeting control siRNA. ( C , D ) The level of viral protein was determined by SDS-PAGE and Western blotting 24 h after the start of infection with YFV ( C ) or WNV ( D ). The band signal is normalized to the loading control βActin) and is expressed as percent of protein expression in the sample transfected with non-targeting control siRNA. ( E ) A549 cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected 48 h later with YFV at an MOI of 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells in the sample and normalized to cells transfected with non-targeting control siRNA. ( F ) HeLa cells were transfected with siRNA (pool or individual oligos) targeting RPS19, RPS3, RPS25, RACK1 or ATP6V1B2 and infected 48 h later with YFV at an MOI 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells and normalized to cells transfected with non-targeting control siRNA. Data are represented as mean ± SD of at least three ( A – C , E , F ) or two ( D ) independent experiments, at the exception of the control data obtained with individual siRNA against ATP6V1B2. Significance was calculated using a one-way ANOVA test of comparisons to control siRNA-transfected samples.

    Journal: Viruses

    Article Title: Uncovering Flavivirus Host Dependency Factors through a Genome-Wide Gain-of-Function Screen

    doi: 10.3390/v11010068

    Figure Lengend Snippet: Ribosomal protein L19 (RPL19) is required for YFV and WNV viral protein synthesis. ( A ) HeLa cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected 48 h later with YFV, WNV, or ZIKV at an MOI of 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells and normalized to cells transfected with non-targeting control siRNA. ( B ) HeLa cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected with YFV, WNV, or ZIKV 48 h later at an MOI of 1. The relative amounts of cell-associated viral RNA were determined by qPCR analysis at 24 h post-infection. Amounts of viral RNA are expressed as genome equivalents (GE) per μg of total cellular RNA and normalized to the sample transfected with non-targeting control siRNA. ( C , D ) The level of viral protein was determined by SDS-PAGE and Western blotting 24 h after the start of infection with YFV ( C ) or WNV ( D ). The band signal is normalized to the loading control βActin) and is expressed as percent of protein expression in the sample transfected with non-targeting control siRNA. ( E ) A549 cells were transfected with siRNA targeting RPL19 or ATP6V1B2 and infected 48 h later with YFV at an MOI of 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells in the sample and normalized to cells transfected with non-targeting control siRNA. ( F ) HeLa cells were transfected with siRNA (pool or individual oligos) targeting RPS19, RPS3, RPS25, RACK1 or ATP6V1B2 and infected 48 h later with YFV at an MOI 1. The percentages of cells expressing the viral E protein were determined by flow cytometry analysis 24 h post-infection. Results are expressed as percent of E-positive cells and normalized to cells transfected with non-targeting control siRNA. Data are represented as mean ± SD of at least three ( A – C , E , F ) or two ( D ) independent experiments, at the exception of the control data obtained with individual siRNA against ATP6V1B2. Significance was calculated using a one-way ANOVA test of comparisons to control siRNA-transfected samples.

    Article Snippet: A549, HeLa, HEK293T and BHK-21 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (Thermo Fischer Scientific, Waltham, MA, USA).

    Techniques: Transfection, Infection, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

    FUS/TLS accumulation at sites of UVA laser-induced damage is dependent on PAR synthesis. ( A ) Human A549 cells were transfected with GFP-hFUS and microirradiated with UVA. Cells were pretreated with vehicle (DMSO) or 500 nM KU58948 1 h before microirradiation. A representative experiment is shown with quantification of GFP-FUS recruitment (mean ± SEM > 30 cells) plotted graphically (bottom left). ( B ) U2OS cells mock-treated or pretreated with 1 µM KU58948 (PARPi) were microirradiated as described above, fixed and immunolabelled for endogenous FUS (middle row) and γH2Ax (bottom).

    Journal: Nucleic Acids Research

    Article Title: PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

    doi: 10.1093/nar/gkt835

    Figure Lengend Snippet: FUS/TLS accumulation at sites of UVA laser-induced damage is dependent on PAR synthesis. ( A ) Human A549 cells were transfected with GFP-hFUS and microirradiated with UVA. Cells were pretreated with vehicle (DMSO) or 500 nM KU58948 1 h before microirradiation. A representative experiment is shown with quantification of GFP-FUS recruitment (mean ± SEM > 30 cells) plotted graphically (bottom left). ( B ) U2OS cells mock-treated or pretreated with 1 µM KU58948 (PARPi) were microirradiated as described above, fixed and immunolabelled for endogenous FUS (middle row) and γH2Ax (bottom).

    Article Snippet: PARP-1+/+ and PARP-1− / − spontaneously immortalized mouse embryonic fibroblasts (MEFs; , ) were grown in MEM +10% FCS. siRNA treatment was conducted on human A549 cells using siGENOME smartpool hFUS siRNA (Thermo Scientific M-009497) or a scrambled siRNA control and Metafectene Pro (Biontex) according to the manufacturer’s instructions.

    Techniques: Transfection

    FUS/TLS accumulates at sites of UVA laser-induced oxidative chromosomal damage. ( A ) A549 cells mock-treated (‘−siRNA’) or pretreated with FUS siRNA (‘+siRNA’) were microirradiated, fixed 2 min later and immunostained for endogenous FUS with anti-FUS antibody (top two panels). A western blot confirming siRNA-mediated knockdown is shown (right). The dotted box in the ‘+siRNA’ samples denotes the position of the UVA laser track. ( B ) U2OS cells were treated as described above and immunostained with anti-FUS (middle row), or anti-γH2Ax antibody (bottom row) as a marker of DNA breaks.

    Journal: Nucleic Acids Research

    Article Title: PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

    doi: 10.1093/nar/gkt835

    Figure Lengend Snippet: FUS/TLS accumulates at sites of UVA laser-induced oxidative chromosomal damage. ( A ) A549 cells mock-treated (‘−siRNA’) or pretreated with FUS siRNA (‘+siRNA’) were microirradiated, fixed 2 min later and immunostained for endogenous FUS with anti-FUS antibody (top two panels). A western blot confirming siRNA-mediated knockdown is shown (right). The dotted box in the ‘+siRNA’ samples denotes the position of the UVA laser track. ( B ) U2OS cells were treated as described above and immunostained with anti-FUS (middle row), or anti-γH2Ax antibody (bottom row) as a marker of DNA breaks.

    Article Snippet: PARP-1+/+ and PARP-1− / − spontaneously immortalized mouse embryonic fibroblasts (MEFs; , ) were grown in MEM +10% FCS. siRNA treatment was conducted on human A549 cells using siGENOME smartpool hFUS siRNA (Thermo Scientific M-009497) or a scrambled siRNA control and Metafectene Pro (Biontex) according to the manufacturer’s instructions.

    Techniques: Western Blot, Marker

    GFP-tagged FUS/TLS accumulates at sites of UVA laser-induced oxidative chromosomal damage. ( A ) Recruitment of eGFP-hFUS to sites of laser damage. Transiently transfected A549 cells were subjected to UVA laser microirradiation along the line indicated. Images were taken at the times (seconds) shown after microirradiation. The graph shows the average GFP fluorescence across six individual experiments and over 100 cells ± SEM. ( B ) Recruitment of eGFP-mFUS to sites of laser damage. Experiments were carried as described in (A). Graph shows the mean of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

    doi: 10.1093/nar/gkt835

    Figure Lengend Snippet: GFP-tagged FUS/TLS accumulates at sites of UVA laser-induced oxidative chromosomal damage. ( A ) Recruitment of eGFP-hFUS to sites of laser damage. Transiently transfected A549 cells were subjected to UVA laser microirradiation along the line indicated. Images were taken at the times (seconds) shown after microirradiation. The graph shows the average GFP fluorescence across six individual experiments and over 100 cells ± SEM. ( B ) Recruitment of eGFP-mFUS to sites of laser damage. Experiments were carried as described in (A). Graph shows the mean of three independent experiments.

    Article Snippet: PARP-1+/+ and PARP-1− / − spontaneously immortalized mouse embryonic fibroblasts (MEFs; , ) were grown in MEM +10% FCS. siRNA treatment was conducted on human A549 cells using siGENOME smartpool hFUS siRNA (Thermo Scientific M-009497) or a scrambled siRNA control and Metafectene Pro (Biontex) according to the manufacturer’s instructions.

    Techniques: Transfection, Fluorescence

    FUS/TLS binds directly to PAR, and a mutation associated with ALS disrupts FUS/TLS recruitment at sites of DNA damage. ( A ) FUS interacts directly with PAR. The indicated amounts of recombinant hFUS or hAPLF were slot blotted onto nitrocellulose membrane and then incubated with (‘+PAR’) or without (−PAR) poly (ADP-ribose). Bound PAR was detected by western blotting. ( B ) Domain structure of FUS/TLS, showing the glutamine/glycine/serine/tyrosine-rich (Q/G/S/Y-rich), glycine-rich (Gly-rich), arginine/glycine-rich (RGG), RNA-binding RRM (RRM), zinc finger (ZF) and nuclear localization (NL) domains. ALS mutations associated with R521 in the NL domain are shown. ( C ) A549 cells transiently transfected with GFP expression construct encoding either wild type (WT) GFP-hFUS or GFP-hFUSR521G were microirradiated with UVA laser and images collected at the indicated times following irradiation. A representative image taken at 90 s following irradiation is shown (right).

    Journal: Nucleic Acids Research

    Article Title: PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

    doi: 10.1093/nar/gkt835

    Figure Lengend Snippet: FUS/TLS binds directly to PAR, and a mutation associated with ALS disrupts FUS/TLS recruitment at sites of DNA damage. ( A ) FUS interacts directly with PAR. The indicated amounts of recombinant hFUS or hAPLF were slot blotted onto nitrocellulose membrane and then incubated with (‘+PAR’) or without (−PAR) poly (ADP-ribose). Bound PAR was detected by western blotting. ( B ) Domain structure of FUS/TLS, showing the glutamine/glycine/serine/tyrosine-rich (Q/G/S/Y-rich), glycine-rich (Gly-rich), arginine/glycine-rich (RGG), RNA-binding RRM (RRM), zinc finger (ZF) and nuclear localization (NL) domains. ALS mutations associated with R521 in the NL domain are shown. ( C ) A549 cells transiently transfected with GFP expression construct encoding either wild type (WT) GFP-hFUS or GFP-hFUSR521G were microirradiated with UVA laser and images collected at the indicated times following irradiation. A representative image taken at 90 s following irradiation is shown (right).

    Article Snippet: PARP-1+/+ and PARP-1− / − spontaneously immortalized mouse embryonic fibroblasts (MEFs; , ) were grown in MEM +10% FCS. siRNA treatment was conducted on human A549 cells using siGENOME smartpool hFUS siRNA (Thermo Scientific M-009497) or a scrambled siRNA control and Metafectene Pro (Biontex) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Recombinant, Incubation, Western Blot, RNA Binding Assay, Transfection, Expressing, Construct, Irradiation

    The synergistic cytotoxic effect of the sequential combination therapy was correlated with the p53 status of the cell A. Combination index for each CDDP concentration after sequential combination therapy in the p53 wild type cell lines A549, A549-NTC, the p53 deficient cell line A549-920 and the p53 mutant cell line CRL-5908. The supporting data for this figure (Mean IC 50 -values and mean CI) can be found in table 2 . B. Protein expression levels of p53 and its main transcription targets MDM2, p21, PUMA, and BAX after monotherapy with CDDP or 5 μM Nutlin-3 or sequential combination therapy in each cell line. C. Percentage of Annexin V PerCP positive cells after treatment in all cell lines, measured by flowcytometric analysis D. Cell cycles distribution after treatment as previously described in all cell lines. Cells were stained with PI and DNA content was measured by flowcytometric analysis. Cells were divided in 3 groups: G1 phase (2n); S-phase (2n-4n); and G2/M phase (4n). (* p

    Journal: Oncotarget

    Article Title: The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer

    doi:

    Figure Lengend Snippet: The synergistic cytotoxic effect of the sequential combination therapy was correlated with the p53 status of the cell A. Combination index for each CDDP concentration after sequential combination therapy in the p53 wild type cell lines A549, A549-NTC, the p53 deficient cell line A549-920 and the p53 mutant cell line CRL-5908. The supporting data for this figure (Mean IC 50 -values and mean CI) can be found in table 2 . B. Protein expression levels of p53 and its main transcription targets MDM2, p21, PUMA, and BAX after monotherapy with CDDP or 5 μM Nutlin-3 or sequential combination therapy in each cell line. C. Percentage of Annexin V PerCP positive cells after treatment in all cell lines, measured by flowcytometric analysis D. Cell cycles distribution after treatment as previously described in all cell lines. Cells were stained with PI and DNA content was measured by flowcytometric analysis. Cells were divided in 3 groups: G1 phase (2n); S-phase (2n-4n); and G2/M phase (4n). (* p

    Article Snippet: Cell lines The NSCLC adenocarcinoma cell lines used in this study were the parental p53 wild type A549 cell line (p53 WT, ECACC, Salisbury, England), and its isogenic derivatives A549-NTC (non-template control, p53 wild type) and A549-920 (p53 shRNA, lentiviral vector) obtained after transduction using the GIPZ lentiviral shRNA VGH5526-EG7157 viral particle set (Thermoscientific, Waltham, USA).

    Techniques: Concentration Assay, Mutagenesis, Expressing, Staining

    The response to Nutlin-3 monotherapy was strongest in the presence of wild type p53 A. Survival curve after 24 hours of treatment with Nutlin-3 (0-50 μM) in the p53 wild type cell lines A549 and A549-NTC, the p53 deficient cell line A549-920 and p53 mutant cell line CRL-5908. The corresponding IC 50 -values are presented as mean ± SD in the figure. B. Protein expression levels of p53 and its main transcription targets MDM2, p21, PUMA, and BAX after treatment with 0, 5, 10 or 25 μM Nutlin-3 in all cell lines. C. Percentage of Annexin V PerCP positive cells after 0, 5, 10 or 25 μM Nutlin-3 in all cell lines. D. Cell cycle distribution after Nutlin-3 monotherapy, Cells were stained with Propidium Iodide and DNA content was measured by flowcytometric analysis. Cells were divided in 3 groups: G1 phase (2n); S-phase (2n-4n); and G2/M phase (4n). (* p

    Journal: Oncotarget

    Article Title: The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer

    doi:

    Figure Lengend Snippet: The response to Nutlin-3 monotherapy was strongest in the presence of wild type p53 A. Survival curve after 24 hours of treatment with Nutlin-3 (0-50 μM) in the p53 wild type cell lines A549 and A549-NTC, the p53 deficient cell line A549-920 and p53 mutant cell line CRL-5908. The corresponding IC 50 -values are presented as mean ± SD in the figure. B. Protein expression levels of p53 and its main transcription targets MDM2, p21, PUMA, and BAX after treatment with 0, 5, 10 or 25 μM Nutlin-3 in all cell lines. C. Percentage of Annexin V PerCP positive cells after 0, 5, 10 or 25 μM Nutlin-3 in all cell lines. D. Cell cycle distribution after Nutlin-3 monotherapy, Cells were stained with Propidium Iodide and DNA content was measured by flowcytometric analysis. Cells were divided in 3 groups: G1 phase (2n); S-phase (2n-4n); and G2/M phase (4n). (* p

    Article Snippet: Cell lines The NSCLC adenocarcinoma cell lines used in this study were the parental p53 wild type A549 cell line (p53 WT, ECACC, Salisbury, England), and its isogenic derivatives A549-NTC (non-template control, p53 wild type) and A549-920 (p53 shRNA, lentiviral vector) obtained after transduction using the GIPZ lentiviral shRNA VGH5526-EG7157 viral particle set (Thermoscientific, Waltham, USA).

    Techniques: Mutagenesis, Expressing, Staining