phprt drgfp  (Addgene inc)

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) Workflow diagram depicting the integrative strategy for identifying GBM-associated lncRNAs and designing CRISPRi sgRNA library. ( B ) Schema of the workflow for construction of lentiviral vectors encoding sgRNA library and experimental design of CRISPRi screens. The scatterplot showing the statistical significance −log 10 ( P value) and log 2 (fold change) [log 2 (FC)] between D21 and D0, for the representative negatively selected sgRNA of the corresponding lncRNA genes in ( C ) U87 and ( D ) U251 cells. The lncRNA genes with zero, one, and at least two significantly depleted sgRNAs are colored in gray, blue, and yellow in the indicated cell lines, respectively. The lncRNA genes with at least two significantly depleted sgRNAs in both cell lines are shown in red. ( E ) Venn diagram showing the overlap of the lncRNA genes with at least two significantly negatively selected targeting sgRNAs in U251 and U87.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques:

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) RT-qPCR analysis of knockdown efficiency for the indicated sgRNAs targeting DARS1-AS1 , RPPH1 , and LINC00944 compared with the negative control sgRNA (sg-NC) in U251 and U87 with stable expression of dCas9-KRAB fusion protein, where GAPDH was used as an internal control. The growth of U251 cells with stable expression of dCas9-KRAB transduced with sg-NC/sgRNAs targeting ( B ) DARS1-AS1 , ( C ) RPPH1 , or ( D ) LINC00944 was monitored (OD 450 absorbance for WST-8 formazan) every 24 hours with CCK-8 assay for 96 hours. ( E ) Growth of the U251 cells transduced with the negative control shRNA (sh-NC)/ DARS1-AS1 –targeting shRNAs was measured with CCK-8 assay for the indicated time intervals. ( F ) Boxplots showing DARS1-AS1 expression in GBM tumors and normal brain tissues based on TCGA and GTEx RNA-seq data. The statistical significance of difference was assessed by Wilcoxon rank sum test. ( G ) Representative pictures of clonogenic growth and ( H ) the bar graph quantifying the colonies formed by U251, U87, and LN229 cells transduced with sh-NC or shRNAs targeting DARS1-AS1 , after cells were cultured for 2 weeks. The growth of ( I ) U251 and ( J ) U87 cells transduced with an empty lentiviral lincXpress vector (Materials and Methods) control (Lv-empty) or DARS1-AS1 overexpression lincXpress vector (Lv- DARS1-AS1 ) was monitored every 24 hours with CCK-8 assay for 96 hours. ( K ) Representative pictures of clonogenic growth and ( L ) the bar graph quantifying the colonies formed by U251 or U87 cells transduced with Lv-empty or Lv- DARS1-AS1 , after cells were cultured for 2 weeks. Data in (A) to (E) and (H) are shown as mean ± SD ( n = 3). ** P < 0.01 by one-way ANOVA with Dunnett’s multiple comparison test. Data in (I), (J), and (L) are shown as mean ± SD ( n = 3). ** P < 0.01 or * P < 0.05 by Student’s t test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Quantitative RT-PCR, Knockdown, Negative Control, Expressing, Control, Transduction, CCK-8 Assay, shRNA, RNA Sequencing, Cell Culture, Plasmid Preparation, Over Expression, Comparison

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) RT-qPCR analysis of RNA-level DARS1-AS1 expression in the immortalized NHAs, the LGG cell lines (Hs683 and SW1783), the immortalized human neural progenitor cell line (ReNCell), GBM cells (U87, U251, and LN229), and GSCs (GSC11, GSC17, GSC20, GSC272, and GSC295). ( B ) The knockdown efficiency of the indicated DARS1-AS1 –targeting shRNAs compared with the negative control shRNA (sh-NC) was determined by RT-qPCR in GSC11, GSC17, GSC20, GSC272, and GSC295 cells. The growth of ( C ) GSC11, ( D ) GSC17, and ( E ) GSC272 cells transduced by the negative control shRNA (sh-NC) or individual DARS1-AS1 –targeting shRNAs was measured by CCK-8 assays every 2 days for 6 days. ( F ) Percentages of GSC cells transduced with sh-NC or DARS1-AS1 –targeting shRNAs that can form neurospheres from single cells determined by self-renewal assay. The Kaplan-Meier survival curves of the mice ( P < 0.0001, log-rank test) with intracranial injection of patient-derived ( G ) GSC11 and ( H ) GSC17 cells stably expressing sh-NC (10 GSC11 mice, 9 GSC17 mice, blue), sh- DARS1-AS1 #1 (10 GSC11 mice, 9 GSC17 mice, red), and sh- DARS1-AS1 #2 (10 GSC11 mice, 10 GSC17 mice, green). ( I ) Thirty (or 21) days after GSC11 (or GSC17) cells stably expressing sh-NC or sh- DARS1-AS1 #1/sh- DARS1-AS1 #2 were intracranially grafted into athymic nude mice, the mouse brains were harvested, fixed, embedded, and stained by H&E. Representative images of H&E-stained tumor section are shown. Scale bar, 5 mm. Tumor volumes were calculated as indicated in Materials and Methods for ( J ) GSC11 and ( K ) GSC17 cells expressing sh-NC (8 GSC11 mice, 10 GSC17 mice, blue), sh- DARS1-AS1 #1 (10 GSC11 mice, 9 GSC17 mice, red) or sh- DARS1-AS1 #2 (10 GSC11 mice, 10 GSC17 mice, green). Data are shown as mean ± SD. ** P < 0.01 or * P < 0.05 by one-way ANOVA with Dunnett’s multiple comparison test. Data in (B) to (F) are shown as mean ± SD ( n = 3). ** P < 0.01 by one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Negative Control, shRNA, CCK-8 Assay, Transduction, Injection, Derivative Assay, Stable Transfection, Staining, Comparison

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) The RNA level of DARS1-AS1 in the nuclear and cytoplasmic fraction of U251 cells was measured by RT-qPCR. MALAT1 RNA and GAPDH mRNA were used as positive controls for nuclear and cytoplasmic fraction, respectively. ( B ) Schematic diagram showing the workflow identifying DARS1-AS1 –associated proteins with MS2bs-tagged RNA affinity purification, coupled with MS analysis. The DARS1-AS1 /protein complex was immunoprecipitated with anti-FLAG antibody in the formaldehyde–cross-linked GBM cells stably expressing MS2bs-tagged DARS1-AS1 and FLAG-tagged MS2 proteins, followed by the MS analysis of the eluted proteins. ( C ) The proteins were retrieved by the pull-down of MS2bs-tagged DARS1-AS1 RNA, and the negative control antisense RNA was visualized by silver staining and subjected to MS analysis. The RNAs retrieved by the RNA pull-down experiments were detected with semiquantitative RT-PCR. ( D ) GSEA analysis of the RNA-seq data generated with siRNA-mediated DARS1-AS1 knockdown revealed enrichment of a YBX1–down-regulated gene signature. NES, normalized enrichment score. ( E ) Boxplots showing the expression of YBX1 in GBM tumors and normal brain tissues based on TCGA and GTEx RNA-seq data. The statistical significance of the expression difference between GBM tumors and normal brain tissues was assessed by Wilcoxon rank sum test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Quantitative RT-PCR, Affinity Purification, Immunoprecipitation, Stable Transfection, Expressing, Negative Control, Silver Staining, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Generated, Knockdown

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) RNA pull-down coupled with Western blot validated the interaction between DARS1-AS1 and YBX1 that was identified from MS analysis. ( B ) RIP with an anti-YBX1/anti-IgG antibody followed by RT-qPCR validated the association of YBX1 with DARS1-AS1 , where anti-IgG antibody was used as a negative control. ( C ) The protein level of YBX1 was determined by Western blot in U251 cells transfected with the negative control siRNA (si-NC) or individual DARS1-AS1 –targeting siRNAs, where β-tubulin was used as a loading control. ( D ) RT-qPCR analysis of YBX1 and DARS1-AS1 RNA level in U251 cells transfected with the negative control siRNA (si-NC) or individual YBX1 -targeting siRNAs. Right: The YBX1 protein expression was determined by Western blot. ( E ) RNA pull-down of the MS2bs-tagged antisense, full-length, and serial deletion mutants of DARS1-AS1 RNA followed by anti-YBX1 Western blotting. The three serial deletion mutants of DARS1-AS1 RNA were generated by deleting 601 to 881, 1 to 300, or 301 to 600 bp, respectively. ( F ) U251 cells stably transduced with the vectors expressing full-length mutant DARS1-AS1 resistant to siRNAs (FL mutant), the deletion mutant with a deletion of 1 to 300 bp (300 del) or the empty vector control, were transfected with the negative control siRNA (si-NC) or siRNAs targeting DARS1-AS1 and were cultured for 4 days. The cell growth was monitored each day with CCK-8 assay. Data in (B) are shown as mean ± SD ( n = 3). ** P < 0.01 by Student’s t test. Data in (D) and (F) are shown as mean ± SD ( n = 3). ** P < 0.01, * P < 0.05, or ns, not significant ( P > 0.05) by one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Western Blot, Quantitative RT-PCR, Negative Control, Transfection, Control, Expressing, Generated, Stable Transfection, Transduction, Mutagenesis, Plasmid Preparation, Cell Culture, CCK-8 Assay, Comparison

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of the genes up-regulated by DARS1-AS1 or YBX1 (i.e., down-regulated by siRNA-mediated depletion of DARS1-AS1 or YBX1 ). ( B ) The bar plot showing the top enriched GO biological processes ranked by –log 10 ( P value) of the GO enrichment analysis of the 421 genes down-regulated by the DARS1-AS1 /YBX1 axis. Flow cytometry–based cell cycle analyses were performed for ( C ) U251 and ( D ) GSC11 cells that were transfected/transduced with the negative control siRNA(si-NC)/shRNA (sh-NC) or siRNA/shRNA targeting DARS1-AS1 and were stained with PI. The percentages of the cells in the indicated cell cycle stages were quantified and shown in the bar graph. RT-qPCR analysis of RNA level of the established regulators of cell cycle, E2F1 , CCND1 , and CCNE2 in ( E ) U251 and ( F ) GSC11 cells transfected/transduced with the negative control si-NC/sh-NC or siRNA/shRNA targeting DARS1-AS1 . The protein level of E2F1 , CCND1 , and CCNE2 was determined by Western blotting in ( G ) U251 and ( H ) GSC11 cells, with/without RNAi-mediated DARS1-AS1 depletion. RT-qPCR analysis of the RNA level of E2F1 , CCND1 , and CCNE2 in ( I ) U251 and ( J ) GSC11 cells transfected/transduced with the negative control si-NC/sh-NC or siRNA/shRNA targeting YBX1 . The protein level of E2F1 , CCND1 , and CCNE2 was determined by Western blotting in ( K ) U251 and ( L ) GSC11 cells, with/without RNAi-mediated YBX1 depletion. Data in (C) to (F), (I), and (J) are shown as mean ± SD ( n = 3). ** P < 0.01 or * P < 0.05 by one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Flow Cytometry, Transfection, Transduction, Negative Control, shRNA, Staining, Quantitative RT-PCR, Western Blot, Comparison

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: RT-qPCR analysis of the RNA level of the known regulators for HR-mediated DSB repair FOXM1 , RAD51 , and BRCA1 in ( A ) U251 and ( B ) GSC11 cells transfected/transduced with the negative control si-NC/sh-NC or individual siRNAs/shRNAs targeting DARS1-AS1 . The protein level of FOXM1 , RAD51 , and BRCA1 as well as the γ-H2AX level was determined by Western blotting in ( C ) U251 and ( D ) GSC11 cells with/without RNAi-mediated DARS1-AS1 depletion. RT-qPCR analysis of the RNA level of HR-mediated DSB repair–related genes FOXM1 , RAD51 , and BRCA1 in ( E ) U251 and ( F ) GSC11 cells transfected/transduced with the negative control si-NC/sh-NC or individual siRNAs/shRNAs targeting YBX1 . The protein level of FOXM1 , RAD51 , and BRCA1 as well as the γ-H2AX level were determined by Western blotting in ( G ) U251 and ( H ) GSC11 cells with/without RNAi-mediated YBX1 depletion. ( I ) Representative pictures of clonogenic growth and ( J ) bar graph quantifying the colonies formed by U251 cells that were transduced with the sh-NC or shRNAs targeting DARS1-AS1 . ( K ) Representative pictures of soft agar colony formation and ( L ) bar graph quantifying the colonies formed in soft agar by GSC11 cells that were transduced with the sh-NC or shRNAs targeting DARS1-AS1 . Scale bar, 500 μm. Data in (A), (B), (E), (F), (J), and (L) are shown as mean ± SD ( n = 3). ** P < 0.01 by one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Quantitative RT-PCR, Transfection, Transduction, Negative Control, Western Blot, Comparison

Journal: Science Advances

Article Title: Multiomics analyses reveal DARS1-AS1 /YBX1–controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance

doi: 10.1126/sciadv.adf3984

Figure Lengend Snippet: ( A ) Genome-wide distribution of the YBX1 eCLIP-seq peaks over different elements. ( B ) The top-ranked 4- or 6-bp-long motif and the top-ranked motif of 8, 10, or 12 bp discovered de novo from the top 2000 YBX1 eCLIP-seq peaks by HOMER (v4.11-2). ( C ) Venn diagram showing the overlap between the 421 protein-coding genes down-regulated by siRNA-mediated knockdown of DARS1-AS1 and YBX1 and the genes whose mRNAs harbored at least one YBX1 eCLIP-seq peak. ( D ) The signal track of the YBX1 eCLIP-seq and the corresponding input were shown for the transcripts of E2F1 , CCND1 , and FOXM1 . RIP with an anti-YBX1/anti-IgG antibody followed by RT-qPCR validated the association of YBX1 with E2F1 , CCND1 , and FOXM1 mRNA in ( E ) U251 and ( F ) GSC11 cells, where anti-IgG antibody was used as a negative control. ( G ) The E2F1 , CCND1 , and FOXM1 mRNA retrieved from the pull-down of MS2bs-tagged DARS1-AS1 RNA, the negative control antisense RNA, or input in U251 cells was detected by semiquantitative RT-PCR. The fold enrichment of the YBX1 RIP signal normalized by the input on E2F1 , CCND1 , and FOXM1 mRNA was determined in ( H ) U251 and ( I ) GSC11 cells with/without RNAi-mediated DARS1-AS1 knockdown. After transcription inhibition with actinomycin D (0 hour), the relative RNA level (normalized by the level at 0 hour) of FOXM1 , E2F1 , and CCND1 in U251 cells ( J to L ) transfected with si-NC/si- YBX1 /si- DARS1-AS1 or GSC11 cells ( M to O ) transduced with sh-NC/sh- YBX1 /sh- DARS1-AS1 at 0, 3, 6, and 9 hours was determined by RT-qPCR. Data in (E), (F), (H), and (I) are shown as mean ± SD ( n = 3). ** P < 0.01 by Student’s t test. Data in (J) to (O) are shown as mean ± SD ( n = 3). ** P < 0.01 by one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To perform DR-GFP reporter assay in U251 cells, U251 cells were transfected with linearized pHPRT-DRGFP (Addgene #26476) and selected with puro (2 μg/μl) for 3 days.

Techniques: Genome Wide, Knockdown, Quantitative RT-PCR, Negative Control, Reverse Transcription Polymerase Chain Reaction, Inhibition, Transfection, Transduction, Comparison