a549 cas9 stable line  (Addgene inc)

Journal: NAR Cancer

Article Title: The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

doi: 10.1093/narcan/zcab002

Figure Lengend Snippet: SCAT7 regulates genome integrity and DNA repair. ( A ) Physical map showing SCAT7 (ELF3-AS1) locus, and underneath the maps showing the RNA-seq tracks for non-treated and cisplatin treated (24 h) WT A549 cells in two biological replicates (wt_NT1–2, wt_Cis1–2). Track height represents normalized read depth. ( B ) Schematic of sub-cellular localization of SCAT7 protein interactors. ( C ) Subcellular localization of SCAT7 lncRNA and control lncRNAs HPRT and MALAT1, determined by cytoplasm-nuclear-chromatin fractionation and RT-qPCR in A549 cell line. Graph shows mean ± SD of two independent experiments. ( D ) RT-qPCR analysis of cisplatin-treated A549 cells in a time course of 48 h shows that SCAT7 is induced along with CDKN1A , the main marker of cisplatin response. ( E ) MTT assay on SCAT7 KD A549 cells with LNAs before and after treatment with cisplatin (24 h). NT = non-treated. Data are represented as percentage compared to untreated cells transfected with negative control. Mean values of at least three independent experiments are shown and statistical significance was derived using a two-tailed unpaired Student's t -test. Data are plotted as mean ± SD (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001). ( F ) qPCR showing the expression of CDKN1A in SCAT7 -depleted cells. ( G ) Western blot showing DNA damage marker γH2A.X in A549 cells transfected with LNA GapmeRs or transduced with shRNA particles. Representative experiments of at least three biological replicates; ( H ) IF images showing γH2A.X levels in the indicated cell lines following SCAT7 KD using LNA-GapmeRs. Scale bar 50 μm. ( I ) SCAT7 expression following treatment with the topoisomerase I and topoisomerase II inhibitors CPT (15 μM) and doxorubicin (5 μM), respectively, in a time course of 48 h. For (D), (F) and (I) experiments were performed in biological triplicates and data are plotted as mean ± SD (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001). Significance was derived using two-tailed unpaired Student's t -test.

Article Snippet: Stable A549 cells were generated using Lentifect™ Purified shRNA lentivirus particles targeting SCAT7 or negative control designed and synthesized by GeneCopoeia™, referred as SCAT7_sh1 and SCAT7_sh2.

Techniques: RNA Sequencing Assay, Fractionation, Quantitative RT-PCR, Marker, MTT Assay, Transfection, Negative Control, Derivative Assay, Two Tailed Test, Expressing, Western Blot, Transduction, shRNA

Journal: NAR Cancer

Article Title: The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

doi: 10.1093/narcan/zcab002

Figure Lengend Snippet: SCAT7 affects DNA repair and replication. ( A ) Western blot analysis of ATR pathway members with indicated antibodies in A549 cells following SCAT7 KD. ( B and C ) Representative IF images of A549 cells transiently or stably transfected with SCAT7 targeting GapmeRs or SCAT7 -sh lentiviral particles following cisplatin treatment. Cells were stained with DAPI (blue) p-ATR (red), γH2A.X and p-CHK-1 (green). Scale bar 50 μm. For (A–C), representative images of at least three independent experiments are shown. ( D ) Quantification of IF experiments depicted in (B) and (C). Data are plotted as average foci number/cell ± SD (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001). Significance was derived using two-tailed unpaired Student's t -test. ( E ) Graphs depicting the percentage of SCAT7 depleted A549 cells and GapmeR CTRL in G0/G1, S and G2/M phase, after exposure to 1.5 mM HU for 5 h. Data are plotted as mean ± SD (* P ≤ 0.05; ** P = 0.01 - 0.001; *** P < 0.001, unpaired two-sided t -test ( n = 3); ( F ) Sketch depicting the experimental design for DNA fiber assay and the examples of fiber tract types representing the different replication structures. SCAT7 KD A549 cells and relative control were sequentially pulse-labeled with 0.1 mM CldU and 0.1 mM IdU for 30 min each. Red tracts, CldU; Green tracts, IdU. ( G ) Replication profiles of SCAT7 KD A549 cells are presented by scoring the percentage of elongating fibers, stalled and newly fired fibers. Five hundred replication signals were scored for each sample. Significance was derived using a two-tailed unpaired Student's t -test. Data are plotted as mean ± SD of two independent experiments (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001). ( H ) qPCR detection of recombined LacZ sequence (LacZ_HR) in SCAT7 KD A549 cells with SCAT7 _LNA1 and LNA2 GapmeRs. SCAT7 KD cells were transfected with LacZ defective dl-1 and dl-2 HR plasmids for 24 h, followed by 24 h 5 μM cisplatin treatment. Data are presented as Relative Quantity (RQ) of cisplatin-treated samples relative to their specific NT control. Data are represented as average RQ of at least six independent replicates ± SD. Dashed horizontal line = NT set as 1. Significance was retrieved using an unpaired Student's t -test (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001).

Article Snippet: Stable A549 cells were generated using Lentifect™ Purified shRNA lentivirus particles targeting SCAT7 or negative control designed and synthesized by GeneCopoeia™, referred as SCAT7_sh1 and SCAT7_sh2.

Techniques: Western Blot, Stable Transfection, Transfection, Staining, Derivative Assay, Two Tailed Test, Labeling, Sequencing

Journal: NAR Cancer

Article Title: The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

doi: 10.1093/narcan/zcab002

Figure Lengend Snippet: SCAT7 regulates TOP1 turnover. ( A ) RIP followed by RT-qPCR for SCAT7 using antibodies against TOP1 and IgG. Data are depicted as fold enrichment on IgG control; ( B ) Western blot of SCAT7 -interacting TOP1 in A549 cells following ChOP using biotinylated antisense probe specific to SCAT7 (SCAT7 as), sense probe (SCAT7 rev), or LacZ negative probe; ( C ) Western blot of TOP1 in transient and stable SCAT7 KD A549 cells following 15 μM CPT treatment for 2 h. Immunoblot images are representative of at least three independent experiments; ( D ) Schematic of the different fragments of SCAT7 transcripts that were inserted into inducible overexpression plasmids; ( E ) Immunoblot of TOP1 in A549 cells transfected with overexpression plasmids containing different fragments of SCAT7 transcript, as described in Figure , after treating cells with 15 μM CPT for 2 h. ( F ) Immunoblot of TOP1–cc levels on DNA following ICE assay in transient and stable SCAT7 KD A549 cells. TOP1–cc levels were normalized to untreated control; ( G ) Western blot of TOP1 in SCAT7 KD A549 cells following CPT treatment in the presence or absence of MG132 proteasome inhibitor. TOP1 levels were normalized to respective untreated control. Immunoblot image is representative of at least three independent experiments; ( H ) Poly-ubiquitin (pUB) immunoblot of immunoprecipitated TOP1 in A549 cells transfected with two LNA GapmeRs targeting SCAT7, and relative control cells following CPT treatment. pUB levels were normalized with respect to CTRL LNA. Immunoblot for the indicated protein (TOP1, γH2A.X and GAPDH) with input (bottom). IgG was used as specificity controls. Representative images of at least three independent experiments; ( I ) Western blot of SCAT7 overexpressing A549 cells following combined treatment with CPT and Cycloheximide (CHX) in a time course of 2 h. Representative images of at least two independent experiments.

Article Snippet: Stable A549 cells were generated using Lentifect™ Purified shRNA lentivirus particles targeting SCAT7 or negative control designed and synthesized by GeneCopoeia™, referred as SCAT7_sh1 and SCAT7_sh2.

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Transfection, Immunoprecipitation

Journal: NAR Cancer

Article Title: The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

doi: 10.1093/narcan/zcab002

Figure Lengend Snippet: SCAT7 depletion in A549/cDDP cells recapitulates the pathways activated by cisplatin in parental cells. ( A ) Phase contrast microscope images of wildtype A549 (left) and A549/cDDP (right) cells. Western blots by the images show the indicated epithelial/mesenchymal markers in A549 and A549/cDDP cells; ( B ) Biological pathways enriched in A549/cDDP cells relative to the parental A549 cells. Graph indicates -log10 FDR for each pathway, the numbers are the percentage of genes enriched from each process. Significance was obtained using P -value ≤ 0.05; ( C ) MTT assay on SCAT7 KD A549/cDDP cells before and after treatment with cisplatin at the IC50 for parental cells. NT refers to non-treated. Data are represented as percentage compared to untreated cells transfected with control LNA. Mean values of at least three independent experiments are shown and statistical significance was derived using a two-tailed unpaired Student's t -test. Data are plotted as mean ± SD (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001); ( D ) SCAT7 expression in A549 WT cells ± 15 μM cisplatin and cDDP cells grown in 15 μM cisplatin shows that SCAT7 is not induced in resistant cells. Data are plotted as mean ± SD of three independent replicates. Statistical significance was derived using a two-tailed unpaired Student's t -test (* P ≤ 0.05; ** P = 0.01 0.001; *** P < 0.001); ( E ) Left: the Venn diagram shows the common genes in the overlapping pathways between cisplatin-treated A549 and SCAT7 KD A549/cDDP cells, and heatmap depicts the log2 FC for the 199 common genes in the two sets of samples. Right: biological pathways enriched in SCAT7 KD A549/cDDP cells. Pathways in bold are common to the ones enriched in parental A549 cells after cisplatin treatment. Graph indicates -log10 FDR for each pathway, the numbers represent the percentage of genes enriched from each process. ( F ) qPCR validation of SCAT7 KD A549/cDDP sequencing data using two independent LNA GapmeRs. Experiments were performed in triplicate and data are plotted as mean ± SD.

Article Snippet: Stable A549 cells were generated using Lentifect™ Purified shRNA lentivirus particles targeting SCAT7 or negative control designed and synthesized by GeneCopoeia™, referred as SCAT7_sh1 and SCAT7_sh2.

Techniques: Microscopy, Western Blot, MTT Assay, Transfection, Derivative Assay, Two Tailed Test, Expressing, Sequencing

Journal: NAR Cancer

Article Title: The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

doi: 10.1093/narcan/zcab002

Figure Lengend Snippet: SCAT7 KD reduces tumor growth in cisplatin resistant xenograft models of LUAD. ( A ) Graphical representation of the therapeutic regimen applied to the A549 mice xenografts. X indicates the time points when tumors where measured. ( B ) Graph showing the average tumor growth of parental A549 xenografts (ΔV = Vtx-Vt0) treated with cisplatin, or CTRL or SCAT7 LNA-GapmeRs in combination with cisplatin, and non-treated controls. ( C ) Pictures of Balb/c nude mice A549/cDDP xenografts at day of dissection. Arrows indicate the subcutaneous tumor. ( D ) Top: Graph showing the average tumor growth of A549/cDDP xenografts depicted in C) treated with control or SCAT7 LNA-GapmeRs alone or in combination with cisplatin, cisplatin only and non-treated controls. Significance was derived using a two-tailed unpaired Student's t -test. Bottom: Tumor growth inhibition (TGI) for A549/cDDP subcutaneous Balb/c nude xenografts depicted in (C). For (B) and (D), average tumor growth was calculated with the formula ΔV = Vtx-Vt0. Data are plotted as mean ± SD (* P ≤0.05; ** P = 0.01 0.001; *** P < 0.001).

Article Snippet: Stable A549 cells were generated using Lentifect™ Purified shRNA lentivirus particles targeting SCAT7 or negative control designed and synthesized by GeneCopoeia™, referred as SCAT7_sh1 and SCAT7_sh2.

Techniques: Dissection, Derivative Assay, Two Tailed Test, Inhibition