Fig. 8
Targeting SHCBP1 in combination with low-dose DNA-damaging drugs compromises DNA repair function. NCI-H1299 cells were transfected with control or SHCBP1 siRNA for 24 h and then treated with 5 μM etoposide or corresponding vehicle for 24 h. The cells were collected for proteomic analysis to identify differentially expressed proteins (DEPs) between siCtrl, siSHCBP1, siCtrl + ETOP, siSHCBP1 + ETOP. A Number of DEPs in siSHCBP1 vs. siCtrl, siCtrl + ETOP vs. siCtrl, siCtrl + ETOP vs. siSHCBP1, siSHCBP1 + ETOP vs. siCtrl, siSHCBP1 + ETOP vs. siSHCBP1 and siSHCBP1 + ETOP vs. siCtrl + ETOP groups, respectively. B Histograms representing clusters of orthologous groups (COG) classification in siCtrl + ETOP vs. siCtrl and siSHCBP1 + ETOP vs. siCtrl + ETOP. The identified differential proteins were classified into four categories (“poorly characterized”, “metabolism”, “cellular processes and signaling” and “information storage and processing”) according to the COG analysis. The horizontal axis represents the number of corresponding differential proteins. The clusters of “Chromatin structure and dynamics” and “replication, recombination and repair” in the category of information storage and processing are highlighted in orange. C, D KEGG pathway and GO (Gene Ontology) analysis of biological process (GO-BP) for the up-regulated DEPs between siCtrl + ETOP vs. siCtrl (C) and the down-regulated DEPs between siSHCBP1 + ETOP vs. siCtrl + ETOP (D). E DEPs clustered by their expression pattern across the different treatment group were achieved by applying fuzzy c-means clustering analysis using the R package Mfuzz. Left panel: Six clusters of DEPs with different expression patterns (n = number of DEPs). Right panel: Heatmaps of each cluster with the top two KEGG pathways and GO analyses of molecular function (GO_MF), cell component (GO_CC), biological process (GO_BP) and protein domain are displayed. F, G NCI-H1299 (F) and HeLa cells (G) were subjected to the same treatment as in A-E (24 h siRNA transfection followed by 24 h low-dose ETOP or CDDP exposure), cells were collected for western blot analysis of SHCBP1, phosphorylated-CHK1, phosphorylated-CHK2, DDB2, XLF and RRM2 with GAPDH as internal reference. NCI-H1299 (5 μM ETOP, 10 μM CDDP); HeLa (3 μM ETOP, 5 μM CDDP)