Additional file 1 of CBX2 shapes chromatin accessibility promoting AML via p38 MAPK signaling pathway
Creators: name: Del Gaudio, Nunzio
name: Di Costanzo, Antonella
name: Liu, Ning Qing
name: Conte, Lidio
name: Dell’Aversana, Carmela
name: Bove, Guglielmo
name: Benedetti, Rosaria
name: Montella, Liliana
name: Ciardiello, Fortunato
name: Carafa, Vincenzo
name: Ambrosino, Concetta
name: Tucci, Valeria
name: Conte, Mariarosaria
name: Martens, Joost H. A.
name: Stunnenberg, Hendrik G.
name: Nebbioso, Angela
name: Altucci, Lucia
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Figure 1. CBX2 silencing impairs leukemic cell survival and induces cell death. A Western blot analysis of CBX2 protein levels in ex vivo leukemic blasts and CD34+ cells. Tubulin was used as loading control. Quantitative analysis (densitometry) of WB is shown as fold change value. B Bar plot representation of CBX2 expression in TGCA, blueprint and bloodspot datasets (P ≤ 0.01). C-F WB analysis showing silencing of CBX2 following shCBX2#1 and shCBX2#2 transduction in U937 (C), K562 (D), HL-60 (E), and NB4 (F) cells. ERK1/2 was used as loading control. G and L Proliferation assay with shCBX2(1), shCBX2(2), and shSCR in HL60 (G) and NB4 (L) cells; experiments were performed at 7 days of puromycin selection; error bars show SD of three biological replicates (*P &lt; 0.05, **P ≤ 0.01). H and M CCK8 analysis of shCBX2- and shSCR-transduced HL60 (H) and NB4 (M) cells; experiments were performed at 7 days of puromycin selection; error bars show SD of three biological replicates (*P &lt; 0.05, **P ≤ 0.01). I and N Percentage of PI-positive shCBX2- and shSCR-transduced HL60 (I) and NB4 (N) cells; experiments were performed at 7 days of puromycin selection; error bars show SD of three biological replicates (*P &lt; 0.05, **P ≤ 0.01). Supplementary Figure 2. shCBX2 affects key pathways in AML. A GSEA analysis showing the top differential hallmark gene sets (nominal P &lt; 0.05) associated with shSCR and shCBX2 cells (NES = normalized enrichment score). B Caspase activity assay of shCBX2- and shSCR-transduced U937 cells untreated or treated with 50 μM of caspase-8 or caspase-9 inhibitors; error bars show SD of three biological replicates. C RT-qPCR analysis showing CBX2 expression in three shCBX2#2- and shSCR-transduced primary AML samples (pt#1, pt#2 and pt#3). Experiments were performed at 3 days of puromycin selection; error bars show SD of two biological replicates (*P &lt; 0.05, **P ≤ 0.01). D and E CUT&amp;RUN-qPCR analysis showing enrichment of CBX2 (D) and H3K27me3 (E) at regulatory regions of indicated positive targets belonging to MAPK pathway. No CBX2 enrichment was observed at negative regions. Results were normalized for the input and expressed as fold change compared to rabbit IGG. Error bars indicate SD of three biological replicates (*P &lt;0.05, **P ≤0.01). Supplementary Figure 3. ATAC-seq and transcriptional expression integration analysis. A Venn diagram showing the 223 common genes from comparison between unique regions by ATAC-seq and differentially upregulated (UP_REG DR) genes by RNA-seq in U937 shCBX2 cells. B Venn diagram showing the 334 common genes from comparison between unique regions by ATAC-seq and differentially downregulated (DOWN_REG DR) genes by RNA-seq in U937 shCBX2 cells. C GO enrichment analysis of the 223 common genes. GO biological processes are reported, indicating the number of genes enriched and statistical significance with FDR q-value. Supplementary Figure 4. GO molecular function and KEGG pathway enrichment analysis of 223 common genes. A GO molecular function and KEGG canonical pathway gene sets of the 223 common genes. B Number of genes enriched and statistical significance with FDR q-value are reported.