Journal: Advanced Science

Article Title: CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming

doi: 10.1002/advs.202005047

Figure Lengend Snippet: CHAF1A gene expression and pathway analyses of NB cells and patients. a) Left: overlap of differentially expressed genes (DEGs, |(fc)| > = 1.25, FDR < 0.1) between control (CHAF1A OFF) and CHAF1A‐overexpressing SHEP cells (CHAF1A ON, 96 h) and CHAF1A ‐correlated genes (FDR < 0.1) in patient cohort 1 ( n = 249) and 2 ( n = 648). Right: GO pathway enrichment analysis of the overlapped genes (ranked by −Log 10 FDR, FDR<0.05). b) Work flow of the metabolomics analysis: global metabolomics analysis was performed by GC‐MS and LC‐MS (DiscoveryHD4 platform, Metabolon Inc.) in CHAF1A‐overexpressing SHEP cells (DOX 1 µg mL −1 for 0, 24, and 72 h, n = 5). c) Metabolite enrichment analysis depicts the pathways significantly up‐ and down‐regulated by CHAF1A (DOX 24 h, FDR < 0.25); Benjamini–Hochberg corrected two‐sided homoscedastic t ‐test. d) Left: schematic presentation (redrawn from Gamble et al. [ ⁵² ] ) of the polyamine pathway with metabolite changes in SHEP cells with or without CHAF1A overexpression for 24 h (red = upregulated metabolites, p ≤ 0.05; blue = downregulated metabolites, p ≤ 0.05). Right: polyamine levels in SHEP cells with or without CHAF1A overexpression for 24 h. Data are mean ± SD ( n = 5). e) Targeted polyamine analysis in IMR32 cells with conditional KD of CHAF1A (DOX 1 µg mL −1 for 5 days). Differential metabolites (FDR < 0.25) are presented in the heatmap (yellow = upregulated; blue = downregulated) ( n = 4). f) Polyamine synthetic and catabolic gene expression in SHEP cells with or without CHAF1A overexpression (24 h). Data are mean ± SD ( n = 2); * p < 0.05, ** p < 0.01, *** p < 0.001; two‐sided unpaired t ‐test. g) Polyamine gene expression in patients with high and low CHAF1A expression (average CHAF1A mRNA expression ± 1SD, Figure ) in patient cohorts 1 and 2. Data are mean ± SEM ( n = 44 in cohort 1 and n = 107 in cohort 2); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two‐sided unpaired t ‐test. h) ODC1 activity in SHEP, GIMEN, and NGP cells with or without CHAF1A overexpression (8 h). One unit is defined as the fluorescence change per minute. Data are normalized by the protein amount and presented as the fold change compared to control (mean ± SD, n = 2); * p < 0.05, ** p < 0.01; two‐sided unpaired t ‐test. MTA = 5'‐methylthioadenosine; AdoMet = S ‐(5'‐Adenosyl)‐ L ‐methionine; AdoHyc = S ‐(5′‐Adenosyl)‐ L ‐homocysteine; FC = fold change.

Article Snippet: [ ] Primary antibodies: CHAF1A (1:1000, Abcam, 126 625), Cell Cycle Regulation Antibody Sampler Kit (1:500 or 1:1000, Cell Signaling, 9932), RAS (1:1000, cell signaling, 3965), and MYCN (1:500, cell signaling, 9405), ODC1 (1:400, Novus Biologicals, NBP2‐32887), HA Tag (1:2000, Abcam, ab9110), CypB (1:1000, 20 361, or 1:500, Santa Cruz Biotechnology, 130 626), β ‐actin (1:10000, Sigma, A5316), and GAPDH (1:10 000, Proteintech, 10494‐AP).

Techniques: Gene Expression, Control, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Over Expression, Expressing, Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: The oncogenic properties of the EWSR1::CREM fusion gene are associated with polyamine metabolism

doi: 10.1038/s41598-023-31576-x

Figure Lengend Snippet: Predicted CREM target genes that were significantly changed with a fold change of Log2 ≥ 2 after CREM siRNA treatment in the CHL-1 cell line.

Article Snippet: Primary antibodies used in the western blotting were: mouse monoclonal anti-CREM (1:1000; clone 3B, Novusbio; Littleton, CO, USA), mouse monoclonal anti-CREB (LB9,1:1000, Abcam), mouse monoclonal anti-ODC1 (1:1000; Clone OTI1G6; OriGene), mouse monoclonal anti-Cyclin B1 (D5C10, 1:1000, Cell Signaling Technology; Danvers, MA, USA), mouse anti- proliferating cell nuclear antigen (PCNA) (PC10, 1:2000, Cell Signaling Technology), rabbit anti-Wee1 (1:1000, Cell Signaling Technology), rabbit anti-KIF11 (1:1000, Proteintech), rabbit monoclonal recombinant anti-c-Myc[Y69] (1:1000, Abcam), rabbit polyclonal anti-SAT11:1000, Proteintech) and mouse monoclonal anti-α-Tubulin (B-5-1-2, 1:2500, Sigma-Aldrich; St. Louis, MO, USA) or HRP-conjugated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Abcam) as control for protein loading.

Techniques: Binding Assay, Transduction, Activation Assay, Activity Assay, Transmission Assay, Cell Differentiation, Expressing, Membrane, Migration

Journal: Scientific Reports

Article Title: The oncogenic properties of the EWSR1::CREM fusion gene are associated with polyamine metabolism

doi: 10.1038/s41598-023-31576-x

Figure Lengend Snippet: Effects of CREM and ODC1 silencing on cell lines CHL-1 and PC-3. ( A ) Immunoblot showing effects of CREM knockdown and ODC1 knockdown on ODC1 protein levels in in CHL-1, HEK-293, PC3, and WM164 cell lines. The blots are cropped and original blots can be found in Supplementary information—Original blots Supplementary Figs. and . ( B ) Proliferation assessed with Ki67 after ODC1 siRNA in CHL-1 and PC3 cell lines. Loss of proliferation was 84–22% in CHL-1 and from 69 to 17% in PC3 cells. ( C ) Senescence assessed with SA β‐galactosidase staining after ODC1 knockdown in CHL-1 cell line, an increase from 15 to 71% was seen. ( D ) Wound healing assay to assess migration after ODC1 knockdown in CHL-1 and PC3 cell lines. ( E ) FACS analysis results after ODC1 siRNA treatment for CHL-1 and PC3 cell lines. An increase in G0/G1 in CHL-1cells from 82 to 91% and PC3 in cells from 83 to 95% ( F ) Downstream cell cycle related proteins and their expression measured by immunoblotting after ODC1 knockdown in CHL-1 and PC3 cell lines. The blots are cropped and original blots/replicates can be found in Supplementary information—Original blots Supplementary Figs. , and . ( G ) Total polyamine concentration compared in CHL-1 and PC3 cell lines after CREM and ODC1 knockdown. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 ( t -test).

Article Snippet: Primary antibodies used in the western blotting were: mouse monoclonal anti-CREM (1:1000; clone 3B, Novusbio; Littleton, CO, USA), mouse monoclonal anti-CREB (LB9,1:1000, Abcam), mouse monoclonal anti-ODC1 (1:1000; Clone OTI1G6; OriGene), mouse monoclonal anti-Cyclin B1 (D5C10, 1:1000, Cell Signaling Technology; Danvers, MA, USA), mouse anti- proliferating cell nuclear antigen (PCNA) (PC10, 1:2000, Cell Signaling Technology), rabbit anti-Wee1 (1:1000, Cell Signaling Technology), rabbit anti-KIF11 (1:1000, Proteintech), rabbit monoclonal recombinant anti-c-Myc[Y69] (1:1000, Abcam), rabbit polyclonal anti-SAT11:1000, Proteintech) and mouse monoclonal anti-α-Tubulin (B-5-1-2, 1:2500, Sigma-Aldrich; St. Louis, MO, USA) or HRP-conjugated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Abcam) as control for protein loading.

Techniques: Western Blot, Knockdown, Staining, Wound Healing Assay, Migration, Expressing, Concentration Assay

Journal: Molecular Cell

Article Title: Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence

doi: 10.1016/j.molcel.2019.08.005

Figure Lengend Snippet:

Article Snippet: Odc1 , Thermo Fisher , Mm01964631_g1.

Techniques: Blocking Assay, Labeling, Recombinant, shRNA, Control, Software