CYBB/NOX2 Antibody (NL7) [PE] NBP1-41012PE from Novus Biologicals, a Bio-Techne Brand

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International journal of oncology
April 01 2018

Fig 1: GSEA of dysregulated pathways in MT+/− and MT−/− U937 cell lines. (A) GSEA plots represent decreased expression of gene sets involved in myeloid differentiation-related pathways in MT+/− (MT1-MT3) and MT−/− (MT11-MT13) groups relative to the WT (WT bulk parental, WT1 and WT2) group. NES, P-value and FDRq value (FDR-corrected P-value) are presented. (B) Scatter plot of genes in the myeloid differentiation pathway, as constructed by comparing Log FPKM values between the MT−/− and the WT groups. Black or small grey dots represent genes in the myeloid differentiation pathway and all genes detected by RNA sequencing, respectively; CYBB and CLEC5A are indicated with arrows. (C) FPKM values of CYBB and CLEC5A in RNA-seq data. (D) GSEA of phagosome maturation and phagosome acidification gene sets for the MT+/− and MT−/− groups compared with the WT group. (E) Scatter plots of phagosome maturation (top panel) and phagosome acidification (bottom panel) gene sets for the MT−/− group in which x- and y-axes refer to the Log FPKM of MT−/− and Log FPKM of WT groups, respectively. (F) Six representative pathways associated with the cell cycle, apoptosis and other signaling pathways. NES are highlighted in blue (downregulated) and red (upregulated). CLEC5A, C-type lectin domain family 5, member A; CYBB, cytochrome B-245 β chain; FDR, false discovery rate; FPKM, fragments per kilobase of transcript per million mapped reads; GSEA, Gene Set Enrichment Analysis; MT, mutated; NES, normalized enrichment score; WT, wild-type.
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International journal of oncology
April 01 2018

Fig 2: Validation of RNA-seq data by RT-qPCR and immunoblotting. (A) Expression levels of genes in WT and ASXL1-mutated U937 cells were validated using RT-qPCR. Data are presented as the means ± standard error of the mean (*P<0.05 vs. the WT group, one-way analysis of variance). (B) ASXL1 expression was also validated by RT-qPCR using two primer sets targeted before (ASXL1-probe 1) and after (ASXL1-probe 2) the mutation point. FPKM values from RNA-seq data obtained from individual WT and ASXL1-mutated cell lines are also shown (ASXL1-FPKM). (C) Proteins extracted from WT, MT+/− and MT−/− cells were subjected to immunoblotting to measure CYBB and ACTL8 protein expression levels using GAPDH as a loading control. (D) Histograms of CYBB expression and MFI of CYBB (left panel), and histograms of CLEC5A expression and MFI of CLEC5A (right panel) in WT and MT U937 cells. Data are presented as the means ± standard error of the mean. *P<0.05 vs. the WT group (WTblk, WT1 and WT2), one-way analysis of variance. ACTL8, actin-like 8; ASXL1, additional sex combs-like 1; CACNA2D3, calcium voltage-gated channel auxiliary subunit α2δ3; CLEC5A, C-type lectin domain family 5, member A; CTSG, cathepsin G; CYBB, cytochrome B-245 β chain; FPKM, fragments per kilobase of transcript per million mapped reads; MFI, mean fluorescence intensity; NAIP, NLR family apoptosis inhibitory protein; OXR1, oxidation resistance 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MT, mutated; WT, wild-type; WTblk, wild-type bulk parental.