PMC

a Protein levels of p-ERK, ERK, and c-jun in HCT116 cells after transduction with shRNA-CDCA5 or sh-Ctrl lentiviurs, as determined by Western-blot analysis. b The integrated band density was determined using ImageLab Software, using GAPDH as the internal control. Gene expression is presented as the percentage relative to the sh-Ctrl group (^*P < 0.05). All experiments were performed in triplicate

a Correlation between CDCA5 protein (IHC-based TMA) and patient survival, was analyzed with Kaplan–Meier plots in 92 CRC patients (P < 0.05). b Representative images of high- or low-CDCA5 expression. Magnification at ×40 or ×200. c Survival analysis based on CDCA5 expression in 320 CRC patients (GEO ID: GSE24551) from a public clinical microarray dataset R2 bioinformatic platform (P < 0.05). Survival was analyzed with log-rank test

IHC was performed to detect CACA5 (a) and PCNA (b) expression in tumor sections, and TUNEL assay (c) was used to determine the apoptotic cells in tissues. The representative images of IHC or TUNELA assay (left panel, ×400) and statistical analysis of CDCA5 protein expression (right panel) are shown (^*P < 0.05 vs. sh-Ctrl group). All experiments were performed in triplicate

a Cell cycle distribution was determined by flow cytometric analysis in HCT116 (left panel) and HT-29 cells (right panel) after CDCA5 knockdown. The percentage of cells in the G0/G1, S, and G2/M phases was calculated. ^*P < 0.05 vs. sh-Ctrl. b Western-blot analysis of CDK1 and CyclinB1 in HCT116 cells after transduction with sh-CDCA5 or sh-Ctrl lentivirus. The integrated band density was determined by ImageLab Software, and GAPDH as the internal control (^*P < 0.05). All experiments were performed in triplicate

A xenograft nude mouse model was used to investigate the effect of CDCA5 knockdown on tumor growth. HCT116 or HT-29 cells were transduced with sh-CDCA5 or sh-Ctrl lentivirus, and then injected subcutaneously into BALB/c nude mice. a Average tumor volume from day 5 after injection (lower panel; ^*P < 0.05 vs. sh-Ctrl). b Representative images of fluorescence (upper panel) and GFP intensity (lower panel; ^*P < 0.05 vs. sh-Ctrl) were observed at the end of experiments. c Representative images of tumor (upper panel) was observed and average tumor weight (lower panel) were assessed (^*P < 0.05 vs. sh-Ctrl group)

a CDCA5 mRNA in tissues from 50 CRC patients, as analyzed by q-PCR. GAPDH was used as internal control. b CDCA5 protein in 73 pairs of CRC tissues and adjacent normal tissues, as determined by IHC-based tissue microarray. Image magnification at ×40 or ×200. T: tumor tissues; N: normal tissues. ^*P < 0.05, tumors vs. normal tissues. c, d CDCA5 mRNA in the R2 Bioinformatic Platform (c) and TCGA (d). ^*P < 0.05, tumors vs. normal tissues. e CDCA5 mRNA and f CDCA5 protein in a panel of CRC cell lines and FHC cells, as determined by q-PCR and Western-blot analyses. GAPDH was used as the internal control. n = 3. ^*P < 0.05, vs. FHC cells. Data are shown as mean ± SD

a Western-blot analysis of CDCA5 in HCT116 (left panel) and HT-29 (right panel) cells after transduction with sh-CDCA5 vs. sh-Ctrl lentivirus. The integrated band density was determined using the ImageLab Software, and GAPDH as the internal control (^*P < 0.05). b After transduction with sh-CDCA5 vs. sh-Ctrl lentivirus for 72 h, cell number was counted using trypan blue exclusion (^*P < 0.05, vs. sh-Ctrl). Left: HCT116; Right: HT-29. c Cell viability of HCT116 and HT-29 cells was determined using CCK-8 assay after transduction with sh-CDCA5 vs. sh-Ctrl lentivirus. Data were normalized to viability on day 1 and shown as fold change. ^*P < 0.05 vs. sh-Ctrl. d Cell survival was analyzed by colony formation assay in HCT116 or HT-29 cells after transduction with sh-CDCA5 or sh-Ctrl lentivirus. Representative images of colonies after CDCA5 knockdown are shown. ^*P < 0.05 vs. sh-Ctrl. All experiments were performed in triplicate

a Hoechst staining was performed to determine the cell apoptosis of HCT116 cells after CDCA5 knockdown (×400 magnification). White arrows indicate cells with morphologic changes characteristic of apoptosis. b Annexin V-APC staining followed by flow cytometric analysis in HCT116 cells after CDCA5 knockdown. ^*P < 0.05, vs. sh-Ctrl. c Caspase-3 activity was determined using a colorimetric assay in HCT116 cells after CDCA5 knockdown, ^*P < 0.05, vs. sh-Ctrl. d Protein levels of BAX, Bcl-2, and PARP in HCT116 cells after transduction with sh-CDCA5 vs. sh-Ctrl lentivirus, as determined by Western-blot analysis. The integrated band density was determined using ImageLab Software, and GAPDH as the internal control. ^*P < 0.05 vs. sh-Ctrl. All experiments were performed in triplicate