Journal: The New Phytologist

Article Title: Fol ‐milR1, a pathogenicity factor of Fusarium oxysporum , confers tomato wilt disease resistance by impairing host immune responses

doi: 10.1111/nph.17436

Figure Lengend Snippet: Fol‐ milR1 is exported into tomato host cells during infection. (a) Tomato wilt disease symptoms caused by infection with Fol for 2 wk in susceptible cultivar ‘Moneymaker’ (MM) and resistant cultivar ‘Motelle’ (Mot). (b) Detection of Fol ‐milR1 in treated tomato roots using low molecular weight RNA gel blots. 40 μg of total RNA was separated by electrophoresis on 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) gels and transferred to a nylon N+ membrane. (γ‐32P)ATP‐labelled specific oligonucleotide probe sequences were used for hybridization. The snRNA gene U6 was used as a loading control. No hybridization could be detected for Fol ‐milR3 or Fol ‐milR4 (shown) or the other four Fol ‐milRNAs (data not shown). (c) Fol‐ milR1 expression was confirmed using quantitative real‐time polymerase chain reaction (qRT‐PCR) with specific primers. Asterisks indicate significant difference when compared to the corresponding control plants in the same treatment, according to the Chi‐square test (*, P < 0.05). Error bars represent the SD of three replicates. (d) Fol‐ milR1 was detected in Fol , Fol protoplasts and tomato root protoplasts using qRT‐PCR with specific primers. Asterisks indicate significant difference when compared to the corresponding control plants in the same treatment, according to the Chi‐square test (*, P < 0.05). Error bars represent the SD of three replicates. (e) Relative levels of fungal biomass were presented by ribosomal intergenic spacer region (IGS) amplified from genomic DNA correlates with Fol biomass in Fol , Fol protoplasts and tomato root protoplasts, using quantitative‐PCR with specific primers. Asterisks indicate significant difference when compared to the corresponding control plants in the same treatment, according to a chi‐squared test (*, P < 0.05). Error bars represent the SD of three replicates.

Article Snippet: For high molecular weight RNA gel blots, 40 µg of total RNA was separated on 7 M urea/15% denaturing polyacrylamide gels in Tris/Boric Acid/EDTA (1× TBE) and subsequently transferred to a nylon N+ membrane. miRNA‐specific oligonucleotide probes (Table ) were end‐labeled using (γ‐32P)ATP (cat. no. M0201; New England Biolabs, Ipswich, MA, USA; oligonucleotide probes were labeled according to the manufacturer's recommendations).

Techniques: Infection, Molecular Weight, Electrophoresis, Polyacrylamide Gel Electrophoresis, SDS Page, Hybridization, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Amplification

Journal: Molecular 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

doi: 10.1186/s12943-018-0889-7

Figure Lengend 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 < 0.05; ** P < 0.01

Article Snippet: Briefly, the RIP lysates from A549 cells were incubated with biotin (Bio)-labeled oligonucleotide probes against circPTK2 ( Bio -5’-TTAAACCAACATCTTTTCTGACACAGAGACGGCG-3′, RiboBio, Guangzhou, China) for 2 h at 25 °C.

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

Journal: Molecular 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

doi: 10.1186/s12943-018-0889-7

Figure Lengend 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 : 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 < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Briefly, the RIP lysates from A549 cells were incubated with biotin (Bio)-labeled oligonucleotide probes against circPTK2 ( Bio -5’-TTAAACCAACATCTTTTCTGACACAGAGACGGCG-3′, RiboBio, Guangzhou, China) for 2 h at 25 °C.

Techniques: 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

Journal: Molecular 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

doi: 10.1186/s12943-018-0889-7

Figure Lengend 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 < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Briefly, the RIP lysates from A549 cells were incubated with biotin (Bio)-labeled oligonucleotide probes against circPTK2 ( Bio -5’-TTAAACCAACATCTTTTCTGACACAGAGACGGCG-3′, RiboBio, Guangzhou, China) for 2 h at 25 °C.

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

Journal: Molecular 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

doi: 10.1186/s12943-018-0889-7

Figure Lengend 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 < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Briefly, the RIP lysates from A549 cells were incubated with biotin (Bio)-labeled oligonucleotide probes against circPTK2 ( Bio -5’-TTAAACCAACATCTTTTCTGACACAGAGACGGCG-3′, RiboBio, Guangzhou, China) for 2 h at 25 °C.

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