Fig 1: Dysregulation of miR-135b affected the tumor progression of GC in vivo. BGC823 cells were transfected with miR-135b mimics, miR-135b mimics+CAMK2D plasmid, miR-135b mimics+empty vector. And then, these cells were xenograft subcutaneously into the immune-deficient nude mice (n=5 for each group). (A) Tumor growth curves in xenograft formation assay. (B) The representative images of xenograft tumors dissected from the nude mice. (C) H&E staining (left panel) and IHC staining of Ki-67 (right panel). Scale bar as indicated. (D) Western blot analysis of CAMK2D and EMT-related proteins in the xenograft tumor tissues. (E) Antagomir-135b or antagomir-NC was delivered into mice body by tail vein injection once every three days from day 0 to 6 when the xenograft models established, and tumor growth curves were shown (n=5 for each group). The arrows indicate the days of injection. (F) The representative images of xenograft tumors. Antagomir-135b significantly inhibits the tumor growth of xenograft models compared with antagomir-NC. (G) H&E staining (left panel) and IHC staining of Ki-67 (right panel). Scale bar as indicated. (H) Western blot analysis of CAMK2D and EMT-related proteins in the xenograft tumor tissues from the mice injected with either antagomir-135b or antagomir-NC. GAPDH was used as an internal control. n=6 independent experiments. *P<0.05 vs Control or Antagomir-NC, #P<0.05 vs miR-135b. (I) Schematic representation of protocol used to study the ability of antagomir-135b to inhibit experimental tumor metastasis (top panel), and the representative images of lungs metastatic colonization by GC cells (bottom panel). Antagomir-135b significantly suppressed distant metastatic colonization.
Fig 2: Levels of hub metabolites and proteins. Boxplots show levels of panels (A–G) hub metabolites ADP, D-glucose 1-phosphate, L-aspartic acid, L-dopa, oxoglutaric acid, rotenone, and sorbitol and (H–L) hub proteins ENPP1, CAMK2D, ABCA8, SLC2A1, and NDUFA12 in CAVD and control samples. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. ABCA8, ATP Binding Cassette Subfamily A Member 8, CAMK2D, calcium2 + /calmodulin-dependent protein kinase II delta; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1NDUFA12, NADH:ubiquinone oxidoreductase subunit A12; SLC2A, solute carrier family 2 member 1.
Fig 3: CAMK2D was downregulated in GC tissues and was associated with poor prognosis of GC patients. (A) The expression level of CAMK2D was markedly increased in GC tissues compared with adjacent tissues or normal gastric mucosa in Peking University Cancer Hospital Gastric Cancer Transcriptome Dataset (PUCH dataset) and ACRG dataset (GSE66229). (B) Kaplan-Meier survival analysis of overall survival (left panel) and post progression survival (right panel) obtained from public gene expression datasets. (C) The mRNA level of CAMK2D in 28 paired primary GC tissues and adjacent tissues was detected by qRT-PCR. (D) Western blot analysis for CAMK2D expression in 12 paired GC tissues and adjacent tissues. Student's t tests were performed. (E) H&E staining and IHC staining of Ki-67 or CAMK2D in GC tissues from patients. (F) Survival analysis of overall survival (left panel) and disease-free survival (right panel) in 146 cases of GC patients. The median expression value was used as the cutoff. HR: Hazard ratio.
Fig 4: IP-MS of GFP-tagged µ2 reveals interaction with core components of the U5 spliceosomal snRNP. (A) Validation of the immunoprecipitation of the different µ2-GFP constructions. (B) Summary of the IP-MS results. On the left, the three independent replicates are shown for each construction, alongside the overlap between them. Only proteins identified in two or three independent replicates were selected as hits. On the top right, the number of identified hits for each construction is denoted. On the bottom right, the protein overlap between the different constructions is depicted using a Venn diagram. (C) Validation of the experimental µ2 IP-MS design using the CAMK2D and CAMK2G potential interactors by Co-IP and western blot. (D) Validation of the co-immunoprecipitation of MRV µ2 protein with components of the U5 snRNP by Co-IP and western blot. Input or IP fractions were resolved on SDS-page gels and submitted to a WB against GFP, EFTUD2, PRPF6, PRPF8, SNRNP200, U2AF35 or the loading control actin.
Fig 5: Restoring the expression of CAMK2D interfered with miR-135b-related functions and remodeled EMT process. GC cells were co-transfected with miR-135b mimics and CAMK2D expression plasmid lacking the miR-135b target region. (A) Western blot analysis of CAMK2D expression after 48 hours' co-transfection. (B) The cell proliferation was monitored by the IncuCyte system. (C) The activity of DNA replication was detected by EdU staining. (D) Overexpression of CAMK2D inhibited miR-135b-induced GC cells migration and invasion, as assessed using the transwell assay. Scale bar as indicated. (E) Western blot analysis of EMT-related proteins E-Cadherin, N-Cadherin and Vimentin after 48 hours' co-transfection. GAPDH was used as an internal control. n=6 independent experiments. *P<0.05 vs Control, #P<0.05 vs miR-135b.
Supplier Page from Abcam for Anti-CaMKII delta antibody [EPR13095]