Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: DHX9 impacts on alternative splicing choices in Ewing sarcoma cells. ( A ) RT-qPCR showing the levels of DHX9 transcript in TC-71 ES cells after 48 h of transfection with either siControl (siCtrl) or siDHX9 oligonucleotides. Values are the mean ± SD of three independent experiments, each performed in triplicate. Statistical analysis was performed by Student’s t -test. ( B ) Western blot showing the DHX9 protein levels in TC-71 cells after 48 h of transfection with either control siRNA or siDHX9, normalized to GAPDH expression. Values are the mean ± SD of three independent experiments, each performed in triplicate. Statistical analysis was performed by Student’s t -test. ( C ) Venn diagram showing overlap between transcriptional- and splicing- regulated genes, identified by comparison of siDHX9 versus siCtrl TC-71 ES cells ( P < 5.053e-06). ( D ) Bar graph showing percentages of AS pattern differentially regulated between siDHX9 and siCtrl. ( E ) Bar graph showing Fold enrichment of Gene Ontology categories (GO) of differentially splicing-regulated genes enriched in siDHX9 TC-71 cells. Histograms represent the fold enrichment score and the -log 10 ( P- values).

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Alternative Splicing, Quantitative RT-PCR, Transfection, Western Blot, Control, Expressing, Comparison

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: DHX9 affects alternative splicing choices of ES cells. ( A–H ) Representative images of the PCR analyses for the indicated alternative splicing events differentially regulated between TC-71 cells transfected with either siCtrl or siDHX9. Schematic representation for each event analyzed is depicted above the representative agarose gel. Green and red boxes indicate the regulated exons in siDHX9 compared with control TC-71 cells, either skipped (green) or included (red). Black arrows in the scheme indicate primers used for the PCR analysis of EPB41L2 , TNIP1 , SPAG9 , C2CD5 , CTTN , CLSTN1 , ABCB6 , and KIAA0753 event. The graphs show the densitometric analysis of the ratio between isoforms with included and skipped exons. P -values were determined by Student’s t -test * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 ( n = 3; mean ± SD).

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Alternative Splicing, Transfection, Agarose Gel Electrophoresis, Control

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: Structural features of DHX9-sensitive exons. Box plots representing comparison between up- regulated, down-regulated, reference (not-regulated in our experiment), and constitutive exons for size ( A ), previous intron size ( B ), following intron size ( C ), MaxEnt Donor Score ( D ), MaxEnt Acceptor Score ( E ), and MaxEnt Acceptor -Donor Score ( F ). P -values are measured by Fisher test.

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Comparison

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: Sequence features of DHX9-sensitive exons. ( A ) Number of enriched pentamers in the regulated exons or in the nearby regions (downstream and upstream). ( B ) Most significant sequence motifs in the regions nearby or within DHX9-sensitive exons (adjusted P -value <0.05). Tomtom Motif Comparison tool, ( https://meme-suite.org/meme/tools/tomtom ) identified the AAUUU and UUUUG motifs as binding sites for HNRNPC, HNRNPCL1, and TIA1. ( C ) Z score was calculated by counting the number of exons with a pentamer (upper part) and by counting the number of pentamers in each exon. ( D ) Immunoprecipitation (IP) experiments were performed to evaluate the interaction between DHX9 and the identified splicing factors in panel (C). Immunoprecipitated proteins were separated by Western blot analysis. The interaction of HNRNPC, Sam68, PTB, and TIA1 with DHX9 was evaluated.

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Sequencing, Comparison, Binding Assay, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: DHX9 immuno-depletion affects U2snRNP recruitment on CTTN pre-mRNA. ( A ) Western blot analysis of SF3B, U1-70K, SF3A2, SF3A3, U2AF65, U2AF35, and DHX9 after IP of endogenous proteins from TC-71 nuclear extracts with anti-DHX9 or control mouse IgGs. The experiments were performed at least three times; statistical analysis was performed by Student’s t- test ( P < 0.01 **). ( B ) IP experiments were performed to evaluate the interaction between DHX9 and the splicing factors identified in A after Benzonase treatment (50 U/ml, for 2 h). ( C ) TC-71 cells were treated with DHX9 inhibitor for 48 hours at the indicated concentrations and PCR analyses for the CTTN alternative splicing event was performed. The graphs show the densitometric analysis of the ratio between isoforms with included and skipped exons. P -values were determined by Student’s t -test * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 ( n = 3; mean ± SD). ( D ) DHX9 expression has been evaluated by RT-qPCR. ( E ) In the upper part, scheme of the affinity chromatography assays from TC-71 nuclear extracts using biotinylated RNA corresponding to CTTN exon 11 pre-mRNA and flanking intronic sequences. Pull-down assays of TC-71 nuclear extracts immunodepleted using either purified rabbit IgGs or anti-DHX9 antibody. The RNA pull-down was performed using in vitro transcribed biotinylated RNA corresponding to CTTN exon 6 pre-mRNA and flanking intronic sequences spanning from −79 to +72 nucleotides as a bait. After extensive washes, pulled-down proteins were analyzed by Western blot using antibodies against DHX9, U170K, U2AF65, U2AF35, SF3B1, and SF3A2. On the right, quantification of pull-down results by densitometric analysis. Bars indicate mean values ± SD of three independent experiments. Significance: binding to CTTN pre- mRNA sequences was evaluated using Student’s t -test: ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. ( F ) Quantification of pull-down of U1, U2, U4, and U6 snRNPs by RT-qPCR. Bars indicate mean values ± SD of three independent experiments. Significance: cross-link to CTTN pre- mRNA sequences was evaluated using Student’s t -test: ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. NE = nuclear extracts; and PD = pull down.

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Immunodepletion, Western Blot, Control, Alternative Splicing, Expressing, Quantitative RT-PCR, Affinity Chromatography, Purification, In Vitro, Binding Assay

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: DHX9 knockdown modulates cellular morphology via CTTN alternative splicing. ( A ) Western blot analysis showing CTTN protein isoforms upon DHX9 silencing. Ten micrograms of total protein extracts were loaded in each lane. β-actin content was used as loading control. ( B ) Representative pictures showing cellular morphology after transfection with either siCTRL or siDHX9 oligonucleotides. Phalloidin staining was used to detect filipodia, DAPI was used for DNA staining. ( C ) TC-71 cells were transfected as in panel (A) and migration assay was performed. The crystal violet-stained migrating cells were photographed and counted. ( D ) Values are the mean ± SD of three independent experiments, each performed in triplicate, considering the migration of siCtrl as 100%. Statistical analyses were performed by Student’s t -test. * P ≤ 0.05; ** P ≤0.01; and *** P ≤ 0.001. ( E ) Measurement of tumour xenograft from shCTRL and shDHX9 mice at 28 days after injection. Statistical analysis was performed by Student’s t -test, P- values: **** P ≤ 0.0001. ( F ) Tumour xenografts were collected from shCTRL and shDHX9 mice ( n = 8 for each group) and weighed. Graph represents the mean of the weights ± SD. Statistical analysis was performed by Student’s t -test, P- values: **** P ≤ 0.0001. ( G ) Representative IHC images of Cortactin staining in tumour xenografts originated from shCTRL and shDHX9 cells. Scale bar: 10 μm. ( H ) Western blot analysis for the expression of Cortactin and Tubulin from xenografts formed by shCTRL and shDHX9 TC-71 cells. On the right, densitometric analysis to evaluate full length and Δexon 11 isoforms of Cortactin. Statistical analysis was performed by Student’s t -test, P- values: ** P ≤ 0.01.

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Knockdown, Alternative Splicing, Western Blot, Control, Transfection, Staining, Migration, Injection, Expressing

Journal: Nucleic Acids Research

Article Title: DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells

doi: 10.1093/nar/gkaf068

Figure Lengend Snippet: CTTN alternative splicing affects cell migration. ( A ) PCR analysis to monitor CTTN splicing from TC-71 cells transfected with either control or si CTTN oligonucleotides directed against the isoform 1 of CTTN containing exon 11. ( B ) Migration assay of siCtrl and siCTTN . Crystal violet-stained migrating cells were photographed and counted. ( C ) Histograms represent the mean ± SD of three independent experiments, each performed in triplicate, considering the migration of siCtrl as 100%. Statistical analysis was performed by Student’s t -test. * P ≤ 0.05; ** P ≤0.01; and *** P ≤ 0.001. ( D ) Hypothetical model of DHX9 splicing regulation (upper part) and its impact on cancer cell migration and invasion (lower part).

Article Snippet: To obtain an additional DHX9 silencing, TC-71 cells were transfected with pGFP-V-RS vector control (shCTRL) or pGFP-V-RS_DHX9 vector (shDHX9) (Origene, Cat# TR30013 and Cat# TG316515, respectively), using Lipofectamine 2000 for 72 h. For the overexpression experiments, cells were transiently transfected with either GFP or GFP-DHX9, using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions and harvested after 48 h. When appropriate, TC-71 cells were treated with DHX9 inhibitor (DHX9-IN-1 Medchem Express, HY-156782) at the indicated concentrations for 48 h.

Techniques: Alternative Splicing, Migration, Transfection, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear receptors PPARγ and TR4 for survival.

doi: 10.4049/jimmunol.1103038

Figure Lengend Snippet: FIGURE 1. PPARg, TR4, and LXRa loss of function modulates intracellular H37Ra load. (A) Loss of function (RNAi: 40 nM; knockdown efficiency ∼95%) of PPARg and TR4 reduces and that of LXRa augments intracellular H37Ra load as monitored by CFUs and by monitoring the percentage of dead bacteria in stable knockdown cell lines (shRNA lentiviral par- ticles stably inserted) by flow cytometry (B). CFU counts are plotted as mean 6 SD, and flow cytometry results are plotted as median. (C) Evaluation of M. tuberculosis survival by im- aging experiments. The extent of colocalization of GFP-H37Ra with acidified lysosomes (stained with LysoTracker; Invitrogen) was de- termined in control and stable knockdown cell lines of PPARg, TR4, and LXRa and the double knockdown of PPARg and TR4. Images shown for each group are those obtained for myco- bacteria (GFP-H37Ra), acidified lysosomes (LysoTracker), or a merge of the two (Merge). Original magnification 360. (D) The overlap coefficient (at a scale of 1) of GFP-H37Ra with the LysoTracker for 20 consecutive infected macrophages. The results were verified by four repetitions of the experiments, each of which was conducted in triplicate. Error bars show SD. *p , 0.05, significant differences with H37Ra control, two-tailed Student t test.

Article Snippet: For stable cell lines, cells were infected with short hairpin RNA (shRNA) lentiviral particles containing three to five expression constructs encoding target-specific nucleotide shRNA as per the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Knockdown, Bacteria, shRNA, Stable Transfection, Cytometry, Staining, Control, Infection, Two Tailed Test

Journal: International journal of urology : official journal of the Japanese Urological Association

Article Title: Astrocyte elevated gene-1 promotes invasion and epithelial-mesenchymal transition in bladder cancer cells through activation of signal transducer and activator of transcription 3.

doi: 10.1111/iju.13486

Figure Lengend Snippet: Fig. 1 AEG-1 contributes to the invasiveness of bladder cancer cells. (a) Measurement of AEG-1 mRNA levels in both UMUC3 and T24 cells transfected with indicated constructs by real-time polymerase chain reaction analysis. (b) UMUC3 cells transfected with indicated constructs were cultured for 24–72 h and tested for proliferation using MTT assays. NS, no significance. *P < 0.05. (c,d) Transwell invasion assays. Both UMUC3 and T24 cells transfected with indicated constructs were seeded onto the upper chamber and allowed to invade through Matrigel-coated inserts for 24 h. Left, representative images of invaded cells. A-shRNA, AEG-1-targeting shRNA; C-shRNA, control shRNA. *P < 0.05.

Article Snippet: The AEG-1-expressing plasmid and empty vector (pCMV6Entry) was obtained from Origene (Rockville, MD, USA) and the AEG-1-targeting shRNA plasmid and scrambled shRNA plasmid from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Construct, Real-time Polymerase Chain Reaction, Cell Culture, shRNA, Control

Journal: International journal of urology : official journal of the Japanese Urological Association

Article Title: Astrocyte elevated gene-1 promotes invasion and epithelial-mesenchymal transition in bladder cancer cells through activation of signal transducer and activator of transcription 3.

doi: 10.1111/iju.13486

Figure Lengend Snippet: Fig. 2 AEG-1 promotes EMT in bladder cancer cells. (a) Morphological changes of UMUC3 cells transfected with empty vector or AEG-1- expressing plasmids. Scale bar, 50 lm. (b) Western blot analysis of indicated proteins in UMUC3-AEG-1 and UMUC3-Mock cells with or without treatment with WP1066 (1 lmol/L) or DMSO (vehicle control) 24 h. (c) Western blot analysis of indicated proteins in UMUC3 cells transfected with AEG-1-targeting shRNA (A-shRNA) or control shRNA (C-shRNA). Numbers below the blots indicate fold-change relative to vector- transfected cells.

Article Snippet: The AEG-1-expressing plasmid and empty vector (pCMV6Entry) was obtained from Origene (Rockville, MD, USA) and the AEG-1-targeting shRNA plasmid and scrambled shRNA plasmid from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, shRNA