Journal: Nature

Article Title: Spliceosomal disruption of the non-canonical BAF complex in cancer.

doi: 10.1038/s41586-019-1646-9

Figure Lengend Snippet: Fig. 2 | Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9. a, BRD9 gene structure and protein domains. Inset illustrates the branchpoints used when the poison exon is included (top) or excluded (bottom). Single and double underlining indicates sequence motifs that were subsequently mutated. aa, amino acid. b, PCR with reverse transcription (RT–PCR) analysis of inclusion of the BRD9 poison exon in a minigene (top) or endogenous (bottom) context, following transfection of minigenes with the illustrated mutations into MEL270 cells with doxycycline (dox)-inducible Flag–SF3B1(WT) or

Article Snippet: For the competition assay to evaluate the cellular effect of sgRNA against BRD9 or Brd9, cell lines were transduced with LentiCas9-Blast (Addgene no. 52962) and then single-cell-sorted into 96-well plates.

Techniques: Mutagenesis, Sequencing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Nature

Article Title: Spliceosomal disruption of the non-canonical BAF complex in cancer.

doi: 10.1038/s41586-019-1646-9

Figure Lengend Snippet: Fig. 3 | BRD9 loss perturbs the formation and localization of the ncBAF complex. a, Schematic of non-canonical BAF (ncBAF; left), canonical BAF (cBAF; middle) and polybromo-associated BAF (PBAF; right) complexes5,6. Solidus denotes one of the proteins is present; comma denotes one or more of the proteins are present or that mutually exclusive inclusion of proteins may occur. b, Cross-linking and immunoprecipitation (IP) with IgG or Flag followed by immunoblotting in K562 cells that express 3×Flag–BRD9. Representative images from n = 3 biologically independent experiments. c, Immunoprecipitation with GLTSCR1 or BRG1 antibody followed by

Article Snippet: For the competition assay to evaluate the cellular effect of sgRNA against BRD9 or Brd9, cell lines were transduced with LentiCas9-Blast (Addgene no. 52962) and then single-cell-sorted into 96-well plates.

Techniques: Immunoprecipitation, Western Blot

Journal: Nature

Article Title: Spliceosomal disruption of the non-canonical BAF complex in cancer.

doi: 10.1038/s41586-019-1646-9

Figure Lengend Snippet: Fig. 4 | BRD9 is a therapeutically targetable tumour suppressor in melanoma. a, BRD9 expression (z-score normalized) in TCGA UVM samples with (n = 18) or without (n = 62) SF3B1 mutations. P value calculated by two-sided t-test. b, Tumour volume 49 days after subcutaneous engraftment of Melan-a cells transduced with the indicated shRNAs into SCID mice. n = 16, 16, 16, 14 and 14 tumours per group (left to right). Error bars, mean ± s.d. P values calculated by two-sided t-test. c, Representative mice from b at day 63. d, Survival of SCID mice engrafted with MEL270 cells that express empty vector, full-length wild-type BRD9 or a BRD9 bromodomain-deletion mutant (ΔBD). n = 5 mice per group. P value calculated by log-rank test. e, Tumour volume from experiments shown in d, 21 days after engraftment. n = 10 tumours per group. Error bars, mean ± s.d. P values calculated by two-sided t-test. f, Colony number (left) and representative images (right) of MEL202 cells (SF3B1R625G) without (control) or with (clone 1, clone 2 and clone 3) CRISPR–Cas9- induced disruption of the BRD9 poison exon. Indels are illustrated in Extended Data Fig. 2o. n = 3 biologically independent experiments. Error bars, mean ± s.d. P values calculated by two-sided t-test at day 3 (middle).

Article Snippet: For the competition assay to evaluate the cellular effect of sgRNA against BRD9 or Brd9, cell lines were transduced with LentiCas9-Blast (Addgene no. 52962) and then single-cell-sorted into 96-well plates.

Techniques: Expressing, Transduction, Plasmid Preparation, Mutagenesis, Control, CRISPR, Disruption

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3′-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file : Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.). b - d eEF2K mRNA expression in control and PD striatum ( b ), medial substantia nigra ( b ) and dorsal nucleus of vagus nerve ( d ). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum ( b )- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra ( c )- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus ( d )- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, * p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T ( n = 2) mutation carrying organoids (T-test, * p < 0.05; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Quantitation Assay, Staining, Control, MANN-WHITNEY, Gene Expression, Microarray, Derivative Assay, Cell Culture, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Brain eEF2K expression and activity in transgenic M83 +/+ PD mice. a eEF2K mRNA levels in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS, n = 10) or pre-formed fibrillar (PFF, n = 13) mouse wild type AS. (Mann–Whitney test, *** p < 0.005; error bars indicate Mean ± S.D.). b - c Western blot analysis of p-eEF2 (T56) and p-ASyn (S129) in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS) or pre-formed fibrillar (PFF) mouse wild type AS ( b ), and corresponding densitometry analysis ( c ) ( n = 7/group; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Transgenic Assay, Injection, Saline, MANN-WHITNEY, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.). c Flow cytometry analysis of reactive oxygen species (ROS), measured by DCFDA staining, in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Over Expression, Mutagenesis, Knockdown, Flow Cytometry, Staining