Journal: Communications Biology

Article Title: Deep genomic characterization highlights complexities and prognostic markers of pediatric acute myeloid leukemia

doi: 10.1038/s42003-023-04732-2

Figure Lengend Snippet: a Bubble plots showing significant biological processes positively (red) and negatively (blue) associated with TP53 alterations by GSEA. The color of the bubbles indicates the −log10 (FWER-adjusted P value). NES normalized enrichment score. b TP53 -altered AML cell lines are more BUB1B -dependent, aneuploid, and etoposide-resistant. Gene effect describes how vital a particular gene is when the gene is knocked down in a cell line. A more negative score implies that a cell line is more dependent on that gene. Boxes represent the interquartile ranges and the black line inside the boxes indicates the median. The whiskers show the maximum and minimum except for outliers (circles, more than 1.5 × interquartile range outside of the box) and extremes (asterisk, more than 3 × interquartile range distant). Data were obtained from the DepMap. P values were calculated by the Mann–Whitney U -test. c Confirmation of BUB1B and CIT knockdown by quantitative RT-PCR and immunoblotting after 72 h of siRNA (si) transfection. mRNA levels were normalized to GAPDH . Consistent immunoblotting results were obtained from two experiments. d Effects of BUB1B and CIT knockdown on THP-1 and MOLM-13 proliferation. After 72 h of siRNA transfection, cell proliferation was monitored by CellTiter-Glo assays (RLU) and trypan blue cell counting (Relative cell number). Proliferation was relative to the 72-h post-transfection time point. RLU relative luminescence. e BUB1B knockdown induced apoptosis. Representative flow cytometric analysis of propidium iodide-stained cells after 5 days of BUB1B knockdown. Consistent results were obtained from 4 independent experiments (sub-G1: 40.2, 33.4, 37.1, and 41.1% in THP-1; 4.2, 3.1, 7.0, and 8.6% in MOLM-13. t = 14.98, df = 6, P = 5.6 ×10 −6 by t -test). f BUB1B knockdown induced a pro-apoptotic gene expression signature. Quantitative RT-PCR analysis was performed after 5 days of siRNA transfection. Expression levels were relative to the negative siRNA group. g CRISPR/Cas9 disruption of TP53 in MOLM-13 cells. Two clones showing heterozygous disruptions of TP53 by fragment analysis and Sanger sequencing. h Quantitative RT-PCR indicates comparable BUB1B knockdown in the negative control and CRISPR clones. i Proliferation of the negative control and CRISPR clones after BUB1B knockdown was assessed by CellTiter-Glo assays. Proliferation was relative to the negative siRNA transfection. Data in charts ( c , d , f , h , i ) are expressed as mean ± SE from three independent experiments. The number of values used to calculate the statistics (one-way ANOVA followed by Dunnett’s test in d , f , h , i ) in each group is indicated. For the post hoc Dunnett’s test, the control category was the negative si group ( d , f ) and the negative control CRISPR clone ( i ).

Article Snippet: BUB1B (Hs01084828_m1) and CIT (Hs00294611_m1) mRNA levels were measured by TaqMan gene expression assays.

Techniques: MANN-WHITNEY, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Cell Counting, Staining, Gene Expression, Expressing, CRISPR, Disruption, Clone Assay, Sequencing, Negative Control, Control

Journal: STAR Protocols

Article Title: Generation of cell-type-specific proteomes of neurodevelopment from human cerebral organoids

doi: 10.1016/j.xpro.2022.101774

Figure Lengend Snippet:

Article Snippet: AI06e-SOX2YFP , WiCell , RRID: CVCL_UB71.

Techniques: Recombinant, Knock-Out, Membrane, Gentle, Protease Inhibitor, Mass Spectrometry, Bicinchoninic Acid Protein Assay, TUNEL Assay, Software, Flow Cytometry