%0 Journal Article
%T Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas
%A Alessandra Pagano
%A Ana Iochabel Soares Moretti
%A Francisco Rafael Martins Laurindo
%A Herve Kovacic
%A Percillia Victoria Santos Oliveira
%A Samir Andrade Mendon£¿a
%A Tiphany Coralie De Bessa
%J Archive of "Cell Death & Disease".
%D 2019
%R 10.1038/s41419-019-1402-y
%X a KRas expression and activity in Caco2, HKE3, and HCT116 cells. Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25£¿ng/mL for 10£¿min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q£¿<£¿0.000001) and HCT116 vs. Caco2 (q£¿<£¿0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH (n£¿=£¿3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological). Test t **p£¿<£¿0.01, (n£¿=£¿4); e¨Cg ROS production after 72£¿h of PDIA1 silencing, measured by HPLC analysis of DHE oxidation products. DHE oxidation produces, among many others, two major products: 2-hidroxyethidium (EOH), which is representative of superoxide species and ethidium, representative of other oxidant species. Ctrl negative, si-RNA control;£¿si-PDI, si-RNA against PDIA1 protein. Representative immunoblots of PDI silencing for each cell type Test t *p£¿<£¿0.05; **p£¿<£¿0.01. For Caco2 cells, n£¿=£¿3; HKE3 cells, n£¿=£¿4; HCT116 cells, n£¿=£¿
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374413/