Fig 1: Regulation of Snail protein by OLA1A. Induction of Snail by TGF-ß1 in OLA1-knockdown A549 cells. Cells were transfected with control siRNA (siControl) and OLA1 siRNA (siOLA1) for 48 hours, and then treated with TGF-ß1 (2.5 ng/mL) or without TGF-ß1 (-) for 72 hours. Cell lysates were subjected to Western blot analysis using anti-Snail and anti-OLA1 antibodies. ß-actin was used as a loading control. B. A quantitative analysis of the relative band intensities for Snail is shown. The bars represent mean ± SD values (n = 3).*p < 0.01, as compared with the siControl group without the treatment of TGF-ß1; #p < 0.01, as compared with the siControl group treated with TGF-ß1; ?p < 0.01, as compared with the siOLA1 group without the treatment of TGF-ß1. C. Basal levels of Snail in MDA-MB-231 and HeLa cells with manipulated OLA1 expression. MDA-MB-231 and HeLa cells were stably transfected with a lentiviral vector containing control (Ctl) shRNA or OLA1-specific shRNA (OLA1). Additionally, HeLa cells were transiently transfected with OLA1-YFP and the control (YFP) vectors. The ectopic expression of OLA1 was confirmed by the presence of a higher molecular weight OLA1-YFP band on top of the endogenous OLA1 band. The Western blot was probed with anti-Snail antibody, and re-probed with anti-OLA antibody (to confirm the gene manipulations) and ß-actin antibody (as a loading control). Note that a non-specific band is above the arrow-indicated Snail.
Fig 2: The association of OLA1 mRNA expression with overall survival in patients with lung cancerThe Kaplan-Meier plots were generated by selecting the OLA1 probe (219293_s_at) and the survival of all lung cancer patients (A), and patients with lung adenocarcinoma (B) or squamous carcinoma patients (C). The x-axis indicates the time of follow-up, and the y-axis indicates survival probability. Small vertical tick-marks indicate individual patients whose survival times have been right-censored.
Fig 3: IHC analysis of OLA1 and E-cadherin expression in human lung cancer tissuesComparison of OLA1 and E-cadherin expression within the same tumor tissue by pairing the microphotographs (vertically) taken from the nearby serial sections (200×original magnification). The left two columns are from two cases of squamous cell carcinoma (A) and the right two columns are from two cases of adenocarcinoma (B) Note that the levels of OLA1 staining are largely opposite to the levels of E-cadherin staining. The result of a Spearman's rank correlation test for correlation between OLA1 and E-cadherin expressions among 110 cases of lung cancer is shown in C.
Fig 4: Knockdown of OLA1 inhibited EMT by regulating GSK3ß/Snail pathwayCells were transiently transfected with the control siRNA or OLA1 siRNA, and at 48 hours post-transfection the cells were treated with 2.5 ng/mL TGF-ß1. Cell lysates collected at indicated time points (0, 15 min, 30 min after treatment with TGF-ß1) were subjected to immunoblotting for the levels of GSK3ß total protein and its phosphorylation at Ser9 residue. The efficiency of OLA1-knockdown was evaluated by anti-OLA1 probing. Immunoblotting analysis of Akt, phospho-Akt (Thr308), phospho-smad2 (Ser465/467), phospho-smad3 (Ser423/425), ß-catenin and Snail was also done. The blot was re-probed with anti-ß-actin antibody for equal loading.
Fig 5: Knockdown of OLA1 results in attenuation of TGF-ß1-induced EMT in A549 cellsA. A549 Cells were transfected with OLA1 siRNA (siOLA1) and control siRNA (siControl) for 48 hours, and then incubated with 2.5 ng/mL TGF-ß1 or without TGF-ß1 (-) in DMEM containing 5% FBS for 72 hours. Cell images were taken under an inverted light microscope (200×original magnification). B. Cell lysates were subjected to Western blot analysis with antibodies as indicated. ß-actin was used as a loading control. C. Densitometric analysis of Western blots obtained in B., showing band intensities of OLA1 and E-cadherin normalized by that of ß-actin. Data represent mean ± SD values from 3 independent experiments. * p < 0.01, as compared with the siControl group without the treatment of TGF-ß1.?p < 0.01, as compared with the Control siRNA group with or without the treatment with TGF-ß1.
Supplier Page from MilliporeSigma for Anti-OLA1 antibody produced in rabbit