Journal: Genome Research

Article Title: Pan-cancer multi-omics analysis and orthogonal experimental assessment of epigenetic driver genes

doi: 10.1101/gr.268292.120

Figure Lengend Snippet: CRISPR-Cas9 screen to perform orthogonal assessment of the driver potential of ERGs in EMT. (A) The screening strategy used to identify positive and negative regulators of EMT among ERGs. (B) Western blot analysis of Cas9 expression in A549 lung cancer cells. “Pool” represents a heterogeneous population of transduced and stably Cas9 expressing cells derived from the parental cells. Individual cell clones derived by cloning rings are numbered 1, 2, 5, 6, 7, 8, and 9. Actin beta was used to normalize for equal loading. (C) Validation of the transduction efficiency of the lentiviral CRISPR ERG library 10 d after puromycin selection using FACS compared with uninfected A549 cells. (D) Enrichment of vimentin-positive (VIM+) population analyzed by FACS after CRISPR ERG library transduction at day 14 after puromycin selection. (E) Validation of cell sorting for the enrichment of VIM+ population by FACS based on the fluorescent antibody EPCAM (EPCAM loss is associated with the mesenchymal cell state) of VIM+, vimentin-negative (VIM−), uninfected cell line, and negative control antibody IgG. (F) Confirmation of cell enrichment for VIM+ and VIM− fractions after sorting. FACS-sorted VIM+ and VIM− populations were grown in culture for 2 wk and analyzed by FACS after staining with cadherin 2 (also known as N-cadherin) antibodies. (G) The overlap of the top EMT-associated ERG gRNAs after Illumina MiSeq deep sequencing; the numbers are derived from two statistical methods (DESeq2 and edgeR) at days 14, 21, and 28 after transduction. (H) Heatmap showing the top ERGs based on enriched and depleted gRNAs at days 14, 21, and 28 after transduction compared with day 0. (I) Volcano plot of ERG gRNAs at day 28 after transduction. (J) Expression analysis by qRT-PCR of EMT markers (cadherin 1 [also known as E-cadherin], vimentin, and cadherin 2) on single targeted A549-VimCas9 clones following EP400 loss of function, relative to expression in the parental A549 Vim Cas9 cell line. (*) P < 0.05, indicates results of one-way ANOVA test. Error bars are SEM of n = 2. (K) Representative image of scratch assay performed on the parental cell line and three generated EP400 KO clones at day 0 and after 24 h (left). On the right, a graph plot showing percentage area closure 24 h after the scratch as averaged of at least six areas analyzed for each clone and for the parental cell line. Experiments were performed in duplicates. (L) Transwell migration assay showing increase of migration at 13 h for A549-Vim Cas9 EP400 KO Cl4 compared to the parental cell line. (KO) Knockout; (Cl) clone; (vim) vimentin. (M) An example of network analysis of selected top ERGs (EP400) associated with the EMT population, obtained with the GeneMANIA package. (N,O) The bar plots show the mutation frequency of EMT-specific ERGs (identified in the CRISPR-Cas9 screen) in clinical samples from nonmetastatic (M0) and metastatic (M1) subsets (based on the annotation of TCGA samples).

Article Snippet: Generation of cell clones stably expressing Cas9 A549 Vim RFP cells (ATCC CCL-185EMT) and MCF10A cells (ATCC CRL-10317) were cultured according to the recommendation by ATCC.

Techniques: CRISPR, Western Blot, Expressing, Stable Transfection, Derivative Assay, Clone Assay, Cloning, Biomarker Discovery, Transduction, Selection, FACS, Negative Control, Staining, Sequencing, Quantitative RT-PCR, Wound Healing Assay, Generated, Transwell Migration Assay, Migration, Knock-Out, Mutagenesis

Journal: Genome Research

Article Title: Pan-cancer multi-omics analysis and orthogonal experimental assessment of epigenetic driver genes

doi: 10.1101/gr.268292.120

Figure Lengend Snippet: CRISPR-Cas9 screen to perform orthogonal assessment of the driver potential of ERGs in cancer cell proliferation. (A) The screening strategy used to identify regulators of cell proliferation among ERGs in both A549 and MCF10A cell lines/clones. (B,C, left) Venn diagrams showing the genes associated with significantly enriched (B) or depleted (C) gRNAs in the screens performed on A549 and MCF10A cells using edgeR analysis in CRISPRAnalyzeR. (B,C, right) Heatmaps showing the adjusted P-values of the commonly enriched (B) or depleted (C) gRNAs in both cell lines. Data are presented as –log10 (adjusted P-value). (D) KEGG pathway analysis performed on genes associated with commonly depleted gRNAs (left) and with commonly depleted and enriched gRNAs (right) in both cell lines. All pathways in red show P < 0.05.

Article Snippet: Generation of cell clones stably expressing Cas9 A549 Vim RFP cells (ATCC CCL-185EMT) and MCF10A cells (ATCC CRL-10317) were cultured according to the recommendation by ATCC.

Techniques: CRISPR, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: TGF-β1 and MUC16 collaborate to induce the alveolar epithelial to mesenchymal transition. The A549 cell line transfected with control siRNA(−) or siRNA-MUC16 was stimulated for 48 h ( A – E ) or 72 h ( F ) with 5 ng/mL TGF-β1 to measure MUC16 ( A ), collagen type I ( B ), α-SMA ( C ), Slug ( D ), and Snail ( E ) mRNA expression by real-time PCR and collagen type I protein levels by Western blotting, quantification was performed by densitometry ( F ). Data are expressed as 2 −ΔCt for mRNA levels and relative to β-actin for protein levels. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−).

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

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

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 modulates the effect of TGF-β1 on SMAD3 phosphorylation. ( A ) A549 and ( B ) MRC5 cell lines transfected with control siRNA(−) or siRNA-MUC16 were stimulated for 40 min with TGF-β1 10 ng/mL to measure p-Smad3 and for 48 h with TGF-β1 5 ng/mL to measure β-catenin by Western blot. A549 ( C ) and MRC5 ( D ) cells were stimulated with 10 ng/mL TGF-β1 for 40 min. Total protein was extracted and immunoprecipitated using MUC16 antibody and probed against p-Smad3 and MUC16 by Western blot (representative images are shown). ( E ) A549 cells were stimulated with 10 ng/mL TGF-β1 for 1 h. MUC16 and p-Smad3 co-localisation was analysed by confocal microscopy to generate a two-dimensional cytofluorogram that selected common localised points of both antibodies (white colour). Scale bars: 10 μm. ( F ) Smad Binding Element (SBE) measure following 10 ng/mL TGF-β1 stimulation during 18 h in A549 cells transfected with siRNA(−) or siRNA-MUC16. Data are expressed relative to Smad3 or β-actin protein level (A-D). The results are expressed as means ± SE of three independent experiments performed in triplicate. Student t-test was used. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) + TGF-β1.

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Binding Assay