Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas

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–g 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？=？