Article Figures & Data
Figures
- Figure 1
Knockdown of FBXW7α alters the phenotype of macrophage in response to colon cancer. A) Efficiency of interference plasmid transfection was examined in RAW264.7 macrophages. B-C) FBXW7α siRNA plasmid or the control vector, were introduced into RAW264.7 for 36h, and then the cells were co-cultured with colon-26 cells for 24h, B) In parallel, total RNA was isolated from co-cultured macrophage for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of cyclooxigenase (COX)-2 and nitric oxide synthase 2 (NOS2); C) The cell-free supernatant was collected and analyzed by ELISA against IL6, IL12p40, and tumor necrosis factor-α (TNFα). GAPDH - glyceraldehyde 3-phosphate dehydrogenase, iNOS - inducible nitric oxide synthase, CTL - control group
- Figure 2
Genome-wide sequencing together with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis identifies FBXW7α-regulated genes of macrophage in response to colon cancer. A) the RNA samples prepared in Figure 1B were used to perform the RNA-seq experiment. Heatmap analysis displaying the dis-regulated targets in FBXW7α siRNA transfected macrophage, comparing with the control group. B) Volcano plot displaying the number of genes with significant differences. C) qRT-PCR analysis of validate five up-regulated targets identified by RNA-seq. D) qRT-PCR analysis of validate three down-regulated targets identified by RNA-seq. CTL - control group, siRNA: FBXW7α - interference plasmid transfection group.
- Figure 3
MiR-205 is required in the regulation of tumor-associated macrophages (TAM) polarization by FBXW7α. A-C) FBXW7α siRNA plasmid or control vector, together with miR-205 inhibitor or inhibitor negative control (inhibitor NC), were introduced into RAW264.7 for 36-hours, and then the cells were co-cultured with Colon-26 cells for 24-hours. The cell-free supernatant was collected and analyzed by ELISA against A) IL6, B) IL12p40, and C)TNFα. siRNA: FBXW7α interference plasmid transfection group. NC - negative control group.
- Figure 4
FBXW7α/miR-205 axis regulate tumor-associated macrophages (TAM) polarization through affecting SMAD1 expression. A-B) SMAD1 is a target of miR-205. A) The region of the human SMAD1 3’UTR predicted to be targeted by miR-205. B) HEK-293T cells were transiently co-transfected with luciferase reporter vectors, and either miR-205 mimics or negative control. Luciferase activities were normalized to the activity of Renilla luciferase. (C-D) FBXW7α siRNA plasmid or control vector, together with miR-205 inhibitor or inhibitor negative control (inhibitor NC), were introduced into RAW264.7 for 36-hours, and then the cells were cocultured with Colon-26 cells for 24-hours. C) The mRNA level was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay; and D) protein level was determined by western-blotting assay. (E-G FBXW7α siRNA plasmid or control vector, together with SMAD1 overexpressing plasmid or NC, were introduced into RAW264.7 for 36-hours, and then the cells were cocultured with Colon-26 cells for 24-hours. The cell-free supernatant was collected and analyzed by ELISA against E) IL6, (F) IL12p40 and G) tumor necrosis factor-α (TNFα).
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