Xpression subset of these genes that showedshowed versus manage cells. We additional validated expression of a of a subset of these genes that modest differential expression modifications modifications within the ANXA2 depleted versus by qRTPCR by qRTPCR modest differential expressionin the ANXA2 depleted versus manage cells control cells (Figure 5C ). We observed a 1.5 observed a 1.five fold upregulation of PRDX2 MDAMB231 ANXA2 KO compared (Figure 5C ). We fold upregulation of PRDX2 in HT1080 andin HT1080 and MDAMB231 ANXA2 to control cells (Figure cells We observed a 1.five fold a 1.52 fold induction and down and down KO in comparison to control5C,E).(Figure 5C,E). We observedinduction of TrxRD2, of TrxRD2, regulation of SCARA3 SCARA3 in MDAMB231 ANXA2 depleted compared to handle cells (Figure 5E,F). regulation of in MDAMB231 ANXA2 depleted compared to control cells (Figure 5E,F). We also investigated the expression of ROS connected ROS related proteins. We important variations inside the We also investigated the expression of proteins. We did not observedid not observe considerable expression within the expression of those depleted versus handle cells, with the exception together with the differences of those proteins in ANXA2proteins in ANXA2 depleted versus manage cells,of PRDX2 (Figure 5G). PRDX2 (Figure 5G). Even though there have been modest variations in the expression of exception of Despite the fact that there have been modest differences within the expression of CATALASE and TrxRDCATALASE and TrxRD2 genes in MDAMB231 ANXA2 KD when compared with control cells we did notCancers 2019, 11,eight ofCancers 2019, 11, x8 ofgenes in MDAMB231 ANXA2 KD in comparison with control cells we did notnot detect SCARA3 protein in observe considerable differences at the protein levels. Of note we could observe considerable differences in the protein levels. Of note we couldn’t detect SCARA3 protein in our extracts. our extracts.Figure 5. Evaluation of ROS related genes and proteins in ANXA2 depleted versus handle cancer cells. Figure 5. Analysis of ROS associated genes and proteins in ANXA2 depleted versus control cancer cells. (a) HT1080 ANXA2 KO 1; ANXA2 KO or WT or or MDAMB231 ANXA2 shRNA1; ANXA2 (a) HT1080 ANXA2 KO 1; ANXA2 KO 22 or WT (b) (b) MDAMB231 ANXA2 shRNA1; ANXA2 shRNA2 or ANXA2 scramble cells had been plated in 100 mm plates for 48 h. Following what RNA p-Dimethylaminobenzaldehyde Purity & Documentation extraction was performed employing the RNeasy mini kit (Qiagen, Manchester, UK) as outlined by the manufacturers directions. A panel of 86 ROS dependent genes was analysed utilizing the RTProfilerTM PCR ArrayCancers 2019, 11,9 ofshRNA2 or ANXA2 scramble cells had been plated in one hundred mm plates for 48 h. Just after what RNA extraction was performed making use of the RNeasy mini kit (Qiagen, Manchester, UK) in accordance with the manufacturer’s instructions. A panel of 86 ROS dependent genes was analysed making use of the RT2 ProfilerTM PCR Array Human Oxidative Anxiety (Qiagen, Manchester, UK) in line with the manufacturer’s directions in a LightCycler 96 instrument (Roche, Basel, Switzerland). (c) HT1080 ANXA2 KO 1; ANXA2 KO 2 or WT; (d) HT1080 ANXA2 shRNA1; ANXA2 shRNA2 or ANXA2 scramble; (e) MDAMB231 ANXA2 KO 1; ANXA2 KO 2 or WT; (f) MDAMB231 ANXA2 shRNA1; ANXA2 shRNA2 or ANXA2 scramble cells were plated in 100 mm plates for 48 h. RNA extraction was performed applying the RNeasy mini kit (Qiagen, Manchester, UK) as outlined by the manufacturer instructions. The gene expression was determined by qRTPCR working with the Onestep NZYSpeedy RTqPCR Green kit (Nzytech, Lisbon, Portugal) as outlined by manufacturer’s guidelines. T.