Fig 1: CircRNF13 promotes the progression of PC via PDK3.a The knockdown efficiency of PDK3 was verified at the mRNA and protein levels in SW-1990 cells by qRT–PCR and Western blotting assays. b Knockdown of PDK3 impaired colony formation ability in SW-1990 cells. c Knockdown of PDK3 impaired cell invasion ability in SW-1990 cells (scale bar: 100 μm). d Tube formation ability of HUVECs cultured in medium collected from circRNF13-overexpressing SW-1990 cells transfected with PDK3 siRNAs or their corresponding controls. e Representative IHC images of PDK3 expression in PC tissues and corresponding normal tissues (scale bar: 200 μm, magnification: top 40x, bottom 200x). f IHC scores of PDK3 in 90 cases of PC tissues with corresponding normal tissues. g Kaplan–Meier overall survival analysis of PDK3 expression in patients with PC (N = 90, P < 0.001). h Pearson correlation analysis of the positive correlation between circRNF13 and PDK3 expression (r = 0.800, P < 0.0001). i Graphical illustration demonstrating the circRNF13/miR-654-3p/PDK3 axis in pancreatic cancer. Data represent at least three independent experiments and are presented as the means ± SD. *P < 0.05, **P < 0.01.
Fig 2: CircRNF13 modulates the miR-654-3p target PDK3.a Hierarchical clustering of significantly differentially expressed mRNAs in MIA PaCa-2 cells under normal and hypoxic conditions. b Schematic illustration showing the targets of miR-654-3p. c qRT–PCR was performed to measure gene expression under hypoxia. d PDK3 mRNA and protein levels after PC cells were transfected with miR-654-3p mimics or inhibitor. e Schematic illustration of wild-type and mutant reporter plasmids. f The luciferase activities in MIA PaCa-2 and SW-1990 cells transfected with miR-654-3p mimics or mimics NC. g The effects of circRNF13 and miR-654-3p on the expression of PDK3. h PDK3 levels in xenograft tumors removed from each group. Data represent at least three independent experiments and are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: PDK2 deficiency not only increases the active form of PDH and has anabolic effects on IL-1β-treated chondrocytes but also decreases oxidative stress and cellular senescence.a The expression of chondrogenic marker genes such as Col2 and Aggrecan, catabolic proteases such as Mmp13 and Adamts5, and SASP-related biomarkers such as Il-6 and Vegf was assessed via qRT‒PCR. The results for mRNA expression are displayed as the fold increase in gene expression normalized to Gapdh. Mean ± s.e.m., *P < 0.05, Mann–Whitney U test. n = 4. b The phosphorylated-PDH (p-S293-PDH), PDH-E1, PDK1, PDK2, PDK3, PDK4, Col2, MMP13, Sirt1, NRF2 and HO-1 protein levels in WT and Pdk2 KO chondrocytes after 24 h of IL-1β were assessed by western blot analysis. Western blot band quantification for p-S293-PDH protein levels was normalized to PDH-E1, and the levels of PDK1, PDK2, PDK3, PDK4, Col2, MMP13, Sirt1, NRF2 and HO-1 were normalized to those of β-actin. *P < 0.05, Mann–Whitney U test. n = 3. c Staining with DHE (red), a fluorescent probe for ROS, and 8-oxo-dG (green), a marker for oxidative DNA damage, was conducted in primary chondrocytes from WT and Pdk2 KO mice treated with IL-1β (10 ng/ml) for 24 h. The nuclei of the cells were counterstained with DAPI (blue). Scale bar, 100 μm. DHE and 8-oxo-dG fluorescence-positive cells were quantified and expressed as ratios to DAPI-positive cells. *P < 0.05. Mann–Whitney U test. n = 4. d The relative expression of oxidative stress marker genes such as Hmox and Fth was evaluated via qRT‒PCR. The mRNA expression results are displayed as the fold increase in gene expression normalized to Gapdh. *P < 0.05. Mann–Whitney U test. n = 4. e SA-β-gal staining was conducted on primary chondrocytes treated with IL-1β (10 ng/ml) for the indicated times. Scale bar, 100 μm. β-Gal-positive cells were counted from six different fields from three biological replicates, and the percentages of positive cells were determined. *P < 0.05. Mann–Whitney U test. n = 3.
Supplier Page from Abcam for Anti-PDK3 antibody