Fig 1: NF-κB p65 protein is enriched and activated in MSC-like cells of gastric cancer tissuesA. IKBKE, NF-κB p65 and phospho-NF-κB p65 analysis by immunochemistry in diffuse type of gastric cancer, intestinal type of gastric cancer and gastritis tissues. Representative images were shown. Stroma cells with elongated and spindle shape are recognized as MSC-like cells. Representative cells are indicated by arrows. B. The percentage of the positive cells among these specified cells in two types of gastric cancer tissues and gastritis tissues were presented as columns. C.-I. GC-MSC and GCN-MSC were isolated from gastric cancer tissues and adjacent non-cancerous gastric tissues. C. qRT-PCR of miR-155-5p expression levels in GC-MSC versus GCN-MSC. D. Western blotting analysis of IKBKE, phospho-NF-κB p65 and NF-κB p65 protein in GC-MSC and GCN-MSC. E. Western blotting analysis of phospho-NF-κB p65 in GC-MSC after treated with PDTC. F. Immunofluorescence assay of α-SMA and FAP protein levels in GC-MSC. G. Colony formation assay. H. Migration analysis. I. Invasion assay. Data were presented as Means ±SD.*, P < 0.05.
Fig 2: miR-155-5p inhibition triggers transition of BM-MSC to GC-MSC-like cells via NF-κB p65 targetingA. The position and sequences of the predicted binding sites complementary to the seed region of miR-155-5p in 3′UTR of NF-κB p65 mRNA are shown in black box. Wild type, luciferase reporter vector contains the sequences of the predicted binding sites. Mutant type, mutant the predicted binding sites with the sequence in the box. B. luciferase activity assay. C. Western blotting analysis of NF-κB p65 in miRNA inhibitor or mimics transfected MSCs. D.-H. Knock down of NF-κB p65 in BM-MSC before transfected with miR-155 inhibitor. Scr, Scramble control; si-NF-κB p65, siRNA against NF-κB p65. I.-L., NF-κB p65 ORF, ORF constructs with 3′-UTR (wild type) or 3′ UTR with mutated target site (mutant type) vectors were transfected separately with miR-155-5p mimics in BM-MSC. D. and I. NF-κB p65 protein detection. E. and J. Immunofluorescence assay of α-SMA. F. Colony formation assay. G. and K. Migration analysis. H. and L. Invasion assay. Data were presented as Means ±SD.*, P < 0.05.
Fig 3: IKBKE validated as a target of miR-155-5p is involved in miRNA inhibitor activating NF-κB p65A. Luciferase activity assay. NF-κB p65 binding motif were constructed into pGL3-Basic vector, which were used to test activation of NF-κB. B. Western blotting analysis of phospho-NF-κB p65 in miRNA inhibitor or mimics transfected MSCs. C.-G. BM-MSC were pretreated with NF-κB inhibitor PDTC for two hours, then transfected with miR-155 inhibitor for 48h. H.-J. IKBKE was validated as target gene of miR-155-5p. K.-O. Knock down of IKBKE in BM-MSC before transfected with miR-155 inhibitor. C. Western blotting analysis of phospho-NF-κB p65 after BM-MSC treated with PDTC. D. and L. α-SMA protein levels detection in BM-MSC; E. -M. Colony formation assay. F. and N. Migration analysis. G. and O. Invasion assay. H. The position and sequences of the predicted binding sites complementary to the seed region of miR-155-5p in 3′UTR of IKBKE mRNA are shown in black box. I. luciferase activity assay. J. Western blotting analysis of IKBKE in miRNA inhibitor or mimics transfected MSCs. K, Detection of IKBKE, phospho-NF-κB p65 and NF-κB p65 protein. Data were presented as Means ±SD.*, P < 0.05.
Supplier Page from Sino Biological, Inc. for Human NF-kB p65 Gene ORF cDNA clone expression plasmid