cancer cell line encyclopedia (ccle) rna-seq datasets  (Broad Institute Inc)

Journal: eLife

Article Title: SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

doi: 10.7554/eLife.73913

Figure Lengend Snippet: ( A ) DAC ( S )– 3 and ( R )– 3 structures. ( B ) Cell viability analysis of HAP-1 or U2OS cells treated for 72 h with the indicated concentrations of ( S )- or ( R )–3. ( C ) Cell viability analysis of individual DAC-resistant clones or wild-type HAP-1 treated for 72 hr with the indicated concentrations of ( S )–3. ( D ) List of mutations identified by RNA-seq or targeted sequencing of HSD17B11 in individual DAC-resistant clones. ( E ) Schematic representation of HSD17B11 functional domains. The positions of the identified mutations are indicated in red. The Y185, K189 (indicated in black), and S172 amino acids are critical for catalysis. ( F ) Analysis by immunoblotting of HSD17B11 levels in wild-type HAP-1 and DAC-resistant clones. Ku80 was used as a loading control. The black arrow indicates HSD17B11 position. ( G ) Analysis by immunoblotting of HSD17B11-GFP levels in individual clones of DAC-resistant clone A5 complemented with GFP, wild-type or S172L mutant HSD17B11-GFP. SAF-A and total H2AX were used as loading controls. ( H ) Cell viability analysis of individual clones of DAC-resistant clone A5 complemented with GFP, wild-type or S172L mutant HSD17B11-GFP treated for 72 h with the indicated concentrations of ( S )– 3 . Figure 1—source data 1. Source data related to . The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions. Figure 1—source data 2. Source data related to . The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions.

Article Snippet: The data visualization tool Ordino ( ) was used to compare the RNA expression levels of selected genes in The Cancer Cell Line Encyclopedia RNA-seq dataset ( http://www.broadinstitute.org/ccle ; ).

Techniques: Clone Assay, RNA Sequencing, Sequencing, Functional Assay, Western Blot, Control, Mutagenesis, Membrane

Journal: eLife

Article Title: SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

doi: 10.7554/eLife.73913

Figure Lengend Snippet: ( A ) Cell viability analysis of wild-type HAP-1, DACR clone A4 and AACR clones treated with AAC ( S )– 4 . ( B ) List of mutations identified on RDH11 by RNA-seq of individual AACR clones. ( C ) Schematic representation of RDH11 with, in red, the positions of the mutations identified and, in black, the three amino acids critical for catalysis. TM = single-pass transmembrane domain. ( D ) Structure of all- trans -retinol, a substrate for RDH11. ( E ) Analysis by immunoblotting of RDH11 levels in wild-type HAP-1, in DACR clone A4 and in the different AACR clones. ( F ) Analysis by immunoblotting of RDH11 and HSD17B11 levels in wild-type U2OS or clones inactivated for either HSD17B11, RDH11 or both. ( G ) Cell viability analysis of wild-type U2OS or U2OS clones inactivated for HSD17B11, RDH11, or both and treated with AADC 12 . ( H ) Structure of prostaglandin E2, a substrate of HPGD. ( I ) Analysis by immunoblotting of GFP and HPGD levels in WT U2OS or U2OS KO HSD17B11 stably complemented with GFP or HPGD-GFP. ( J ) Cell viability analysis of U2OS or U2OS inactivated for HSD17B11, stably complemented with either HSD17B11-GFP or HPGD-GFP and treated for 72 h with AllAC ( S , S a )- or ( R , S a )– 5 . Figure 5—source data 1. Source data related to . The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions. Figure 5—source data 2. Source data related to . The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired using autoradiographic films. Two different immunoblotting of the same extracts were used for this figure (respectively labeled upper and lower). The regions used to generate the figure are highlighted for each immunoblot by back squares in the jpg files. Figure 5—source data 3. Source data related to . The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The same extracts were loaded twice (left and right part of the membrane) and the membrane sliced to simultaneously probe GFP and HPGD. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains overlays of the chemiluminescent signal with a picture of the membrane to locate the protein ladder positions.

Article Snippet: The data visualization tool Ordino ( ) was used to compare the RNA expression levels of selected genes in The Cancer Cell Line Encyclopedia RNA-seq dataset ( http://www.broadinstitute.org/ccle ; ).

Techniques: Clone Assay, RNA Sequencing, Western Blot, Stable Transfection, Membrane, Labeling

Journal: eLife

Article Title: SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

doi: 10.7554/eLife.73913

Figure Lengend Snippet: ( A ) List of genes carrying near homozygous non- or mis-sense mutations in each of the seven AACR clones analyzed by RNA-seq. For each gene, the impact on the protein sequence is specified. ( B ) Graphical representation of the genes identified as mutated in each clone. The only gene mutated in more than four clones is highlighted in red. The dashed lines indicate that RDH11 is not expressed in the AACR#B4 clone and carries two heterozygous mutations, T227K and R108* in clone AACR#A6. ( C ) Histogram representing the ratio between the normalized levels (RPKM) of RDH11 mRNA in each mutant clone as compared to the wild-type HAP-1.

Article Snippet: The data visualization tool Ordino ( ) was used to compare the RNA expression levels of selected genes in The Cancer Cell Line Encyclopedia RNA-seq dataset ( http://www.broadinstitute.org/ccle ; ).

Techniques: Clone Assay, RNA Sequencing, Sequencing, Mutagenesis

Journal: eLife

Article Title: STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells

doi: 10.7554/eLife.53003

Figure Lengend Snippet: ( A ) Heatmap of Enrichment Scores (ES) from RNA-seq analysis showing that partial suppression of MAP4K4 expression in HEK TER cells upregulates a transcriptional signature closely resembling four published, independently generated YAP1 signatures and two signatures for YAP1/TAZ from Ingenuity Pathway Analysis (IPA) using Information Coefficient (IC) as a similarity metric. ssGSEA was performed and enrichment scores are represented as indicated in the color bar with red indicating relative enrichment and blue depletion. The three columns in the heatmap represent triplicates for each condition. ( B ) Immunoblot depicting changes in phosphorylation of YAP1 on a key negative regulatory site (S127) following partial MAP4K4 knockdown or expression of MAP4K4 K54R in HEK TER cells. The values below the blot represent quantitation of the YAP1 pSer127 signal relative to the total YAP1 from the immunoblot. ( C ) Immunoblot showing changes in phospo-LATS1 following partial MAP4K4 knockdown in HEK TER cells. Quantification of the LATS1 Thr1079 signal relative to total LATS1 from the immunoblot is shown below the gel. ( D ) Immunoblot depicting changes in the YAP1 target genes CTGF and CYR61 following partial MAP4K4 knockdown and the ratios of the levels of CTGF/β-actin, CYR61/β-actin are shown below the blot. β-actin shown was performed in the same blot. Changes in the mRNA levels of YAP1 target genes CTGF ( E ) and CYR61 ( F ) upon MAP4K4 suppression. ( G ) Immunoblot depicting changes in phosphorylation of YAP1 on S127 following STRN4 knockdown in HEK TER ST. The values below the blot depict quantitation of the YAP1 pSer127 signal relative to total YAP1 from the blot (**p<0.001, ****p<0.00001). Figure 7—source data 1. Quantification of CTGF and CYR61 gene expression (TPM).

Article Snippet: We also generated signatures from these experiments to apply them in the CCLE RNA Seq dataset ( www.broadinstitute.org/ccle ) ( ) using ssGSEA.

Techniques: RNA Sequencing, Expressing, Generated, Western Blot, Phospho-proteomics, Knockdown, Quantitation Assay, Gene Expression

Journal: eLife

Article Title: STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells

doi: 10.7554/eLife.53003

Figure Lengend Snippet: ( A ) Quantification of AI growth obtained following partial MAP4K4 suppression alone or when combined with YAP1 suppression ( shYAP1 ) in HEK TER cells. Transformation induced by partial MAP4K4 suppression depends on YAP1 . ( B ) Quantification of AI growth following STRN4 knockdown with or without co-expression of YAP1 WT or the S5A mutant in HEK TER ST cells. YAP1 rescues the transformation defect of STRN4 suppression by shSTRN4-55 and shSTRN4-58 (immunoblots are shown in ). ( C ) Heatmap of ES depicting YAP1 genesets from the literature significantly associated with the MAP4K4 knockdown signature ES using Information Coefficient (IC) as a similarity metric. ssGSEA was performed using these genesets across the CCLE dataset, and enrichment scores are represented as indicated in the color bar, with red indicating relative enrichment and blue depletion (FDR < 0.0001). ( D ) Heatmap depicting top dependency genes (bottom heatmap) in the Project Achilles dependency profiles that associated with MAP4K4 knockdown signature ES (top heatmap). The top heatmap represents ES from ssGSEA, while the bottom heatmap represent relative dependency (blue indicating strong dependency). Cell lines with low MAP4K4 transcriptional activity (in blue on top) were the most dependent on TEAD1 and TAZ1 (FDR < 0.0001). ( E ) Heatmap depicting co-dependency analysis of MAP4K4 using IC across the Project Achilles data. The genes most significantly associated with MAP4K4 dependency were enriched for the Hippo/YAP1 pathway, as well as components of the STRIPAK complex (All associations FDR < 0.0001 except TAOK2 , SAV1 : FDR = 0.002). (Student’s t-test, *p<0.01, **p<0.001, ****p<0.00001, n.s. = not significant). Figure 8—source data 1. Quantification of AI growth with changes in YAP1 and MAP4K4.

Article Snippet: We also generated signatures from these experiments to apply them in the CCLE RNA Seq dataset ( www.broadinstitute.org/ccle ) ( ) using ssGSEA.

Techniques: Transformation Assay, Knockdown, Expressing, Mutagenesis, Western Blot, Activity Assay