(E) Western blot analysis of FXR1, p21, and PNPT1 in UMSCC11A cells under individual and double KD of FXR1 and PNPT1. in FXR1 KD A549 cells. RNU6 served as an endogenous control. (E) qRT-PCR is usually showing the KD efficiency in shFXR1 (used in Fig 1) treated cells used for miRNA analyses. Actin and Thioridazine hydrochloride GAPDH served as endogenous controls. (F) Western blot showing the KD efficiency of FXR1 by shRNA (TRCN0000158932) compared to a scrambled shRNA where -Actin serves as a loading control. (G) qRT-PCR of altered miRNAs in FXR1 KD (TRCN0000158932) UMSCC74B cells. RNU6 served as an endogenous control. Data from B-D and F-G represent the mean of n? = 3 experiments. Statistical significance (and served as endogenous controls. (B) Western blot analyses showing recombinant FXR1 protein expression before and after dialysis with the anti-His-tag antibody. (C) Western blot analyses showing recombinant FXR1 protein expression before and after dialysis with the anti-FXR1 antibody. (D) Beta-galactosidase assay showing, like the previous observation [34], instead of the miRNA alone, both miR301a-3p and TERC downregulation can induce senescence in UMSCC74B cells.(PDF) pgen.1008580.s002.pdf (11M) GUID:?B7D04DA6-3CF2-4997-B68C-57589C89DAE2 S3 Fig: The stability of miR301a-3p is FXR1 dependent. (A) qRT-PCR assay of FXR1 KD UMSCC11A cells showing significant down- and up-regulation of and did not show any switch after FXR1 KD. Both and served as endogenous controls. (B) Western blot analyses of FXR1, p21, and AGO2 from UMSCC11A cells collected at different time points after FXR1 KD. GAPDH serves as a loading control. (C) qRT-PCR assay of FXR1 KD UMSCC11A cells showing significant miR301a-3p decay from 48 hrs compared to control. Cells were collected at the designated time points after shRNA transduction. RNU6 served as an endogenous control. (D) qRT-PCR assay of FXR1 and AGO2 KD UMSCC74B cells. Unlike FXR1 KD cells, and did not show any biologically relevant changes after AGO2 KD. Both and served as endogenous controls. (E) Western blot analyses of FXR1, p21, and AGO2 from UMSCC74B cells after AGO2 KD. GAPDH serves as a Thioridazine hydrochloride loading control. (F) qRT-PCR assay of AGO2 KD UMSCC74B cells showing no significant regulation of miR301a-3p compared to control at 72hrs of transduction. RNU6 served as an endogenous control. Data here represents the imply of n? = 3 experiments. Statistical significance (in UMSCC11A cells under individual and double KD of FXR1 and PNPT1. Both and served as endogenous controls. (E) Western blot analysis of FXR1, p21, and PNPT1 in UMSCC11A cells under individual and double KD of FXR1 and PNPT1. GAPDH serves as an endogenous control. (F) EMSA shows that both rFXR1 and rPNPT1 proteins are unable to bind and degrade, respectively, the in vitro transcribed miR204-5p. Data here represent the mean of n? = 3 experiments. Statistical significance (mRNA and reduce its expression. (A) qRT-PCR analyses to test the expression of miR301a-3p in UMSCC74A cells treated with miRNA inhibitor with scrambled control. RNU6 served as an endogenous control. (B) p21 protein is up-regulated in miR301a-3p inhibitor transfected UMSCC74A cells. -Actin serves as a loading control. (C) 3UTR luciferase activity is significantly up-regulated in the presence of miR301a-3p inhibitor in UMSCC74A cells compared to the scrambled control transfected cells. Forty-eight hours after Mouse monoclonal to CD32.4AI3 reacts with an low affinity receptor for aggregated IgG (FcgRII), 40 kD. CD32 molecule is expressed on B cells, monocytes, granulocytes and platelets. This clone also cross-reacts with monocytes, granulocytes and subset of peripheral blood lymphocytes of non-human primates.The reactivity on leukocyte populations is similar to that Obs transfection of UMSCC74A cells with miRNA control and 301a-3p inhibitor along with empty 3-UTR luciferase plasmid and wild type 3-UTR, the lysates were analyzed for luciferase activity using a luminometer. The Thioridazine hydrochloride empty 3UTR luciferase plasmid served as a transfection and loading control. Values are the means SD from three independent experiments by using an unpaired two-sample t-test. (D) Expression of miR301a-3p in UMSCC74A cells treated with miRNA mimics. RNU6 served as an endogenous control. (E) p21 protein is down-regulated in miR301a-3p mimic treated UMSCC74A cells. -Actin serves as a loading control. (F) 3-UTR (full-length wild type and mutated miRNA binding sites) luciferase Thioridazine hydrochloride activity with an expression of miR301a-3p mimic in UMSCC74A cells. In the presence of miR301a-3p mimic, the p21 3-UTR luciferase activity significantly reduces whereas the mutants show a highly significant up-regulation. Experiments were performed as described in (C). (G) qRT-PCR analyses to test the expression of miR301a-3p in A549 cells treated with miRNA inhibitor with scrambled control. RNU6 served as an endogenous control. (H) p21 protein is up-regulated in miR301a-3p inhibitor transfected A549 cells. GAPDH serves as a loading control. (I) 3UTR luciferase activity is significantly up-regulated in.