Fig 1: miR-433 inhibits the WNT signaling pathway through TMED5. (A) Western blotting showed the protein levels of p-ß-catenin, ß-catenin, c-myc, cyclin D1 and GAPDH in A549 and H1299 cells transfected with the indicated plasmids. (B) Top/Fop luciferase reporter assay was performed to detect WNT activity. (C) Immunohistochemistry showed the protein expression levels of TMED5 and ß-catenin. Scale bar, 50 µm. (D) Model of potential association of miR-433 and its target gene TMED5. *P<0.05 and **P<0.01 vs. pcDNA3 + pcDNA3. p, plasmid; TMED5, transmembrane p24 trafficking protein 5; miR, microRNA; p, phosphorylated; t, total; Lenti, lentivirusl Con, control.
Fig 2: miR-433 directly targets TMED5. (A) Predicted miR-433 binding sites in TMED5 mRNA WT and MUT using StarBase V3.0. (B and C) EGFP intensity in A549 and H1299 cells co-transfected with pri-miR-433 or ASO-miR-433 and the WT or MUT 3′-UTR of TMED5. (D) TMED5 mRNA expression with the indicated transfections was measured by reverse transcription-quantitative PCR assay in A549 and H1299 cells. (E) TMED5 protein expression in A549 and H1299 cells transfected with pri-miR-433 or ASO-miR-433 and respective controls was determined by western blotting. (F) GEPIA database showing the TMED5 protein expression. (G)TCGA database showing the TMED5 mRNA expression. (H) Human Protein Atlas showing that high protein expression levels of TMED5 predicted a poor prognosis in patients with NSCLC. *P<0.05, **P<0.01 and ***P<0.001 vs. pcDNA3 or ASO-NC. ns, not significant; WT, wild-type; MUT, mutated; 3′-UTR, 3′-untranslated region; miR, microRNA; TMED5, transmembrane p24 trafficking protein 5; NC, negative control; ASO, antisense oligonucleotide; TCGA, The Cancer Genome Atlas; NSCLC, non-small cell lung cancer; R, resistant; Adj, adjacent.
Fig 3: TMED5 functions as an oncogene in NSCLC. Efficiency of pTMED5 was detected by (A) reverse transcription-quantitative PCR and (B) western blot assay in A549 and H1299 cells. (C) Role of altered TMED5 expression on the viability of A549 and H1299 cells was detected by CCK-8 assay. (D) Colony formation assay showed the proliferation ability of A549 and H1299 cells transfected with pTMED5. (E) Flow cytometric analysis showed that TMED5 overexpression in A549 and H1299 cells resulted in a decrease of cells in the G0/G1 phase and an increase of cells in the S and G2 phases. (F) TMED5 overexpression promoted the proliferation index. (G) Flow cytometric assay showed that TMED5 overexpression decreased apoptosis in A549 and H1299 cells. (H) Western blotting showed the protein expression levels of C-caspase 3 and C-PARP in A549 and H1299 cells. (I) Transfected A549 and H1299 cells were treated with 0–4 µg/ml cisplatin in 96-well plates for 24 h. CCK-8 assay was then used to investigate cell viability. (J) Comet assays showed the degree of DNA breaks in A549 and H1299 cells transfected with the indicated plasmids treated with 10 µg/ml taxol for 4 h. (K) Western blotting showed the protein expression levels of 53BP1 and H2AX in A549 and H1299 cells transfected with the indicated plasmids treated with 10 µg/ml taxol for 4 h. (L and M) Deep single cell transcriptome data showed the immune cell infiltration in NSCLC and the control group. These figures were taken form ‘Global characterization of T cells in non-small cell lung cancer by single-cell sequencing’ (http://lung.cancer-pku.cn/index.php). *P<0.05 and **P<0.01 vs. pcDNA3. ns, not significant; p, plasmid; CCK-8, Cell Counting Kit-8; OD, optical density; C, cleaved; PARP, poly(ADP-ribose)polymerase; 53BP1, tumor protein p53 binding protein 1; NSCLC, non-small cell lung cancer; TMED5, transmembrane p24 trafficking protein 5; TPM, transcript per million.
Supplier Page from Abcam for Anti-TMED5/p28 antibody