Fig 1: (a) Combined bisulfite restriction assay (COBRA) results of 4 regions of interest located in SPRY2 from 10 colorectal cancer (CRC) patients (T(tumor)# patient ID number) and adjacent normal colonocytes (C(control)# patient ID number). Each sample was run on two lanes: lane 1) bisulfite-treated PCR amplified DNA without restriction enzyme digestion, which served as a reference control for unmethylated CpG and lane 2) bisulfite-treated PCR amplified DNA digested with a restriction enzyme recognizing amplicons containing a 5′mCpG sequence. + Lane: PCR product treated with restriction enzyme lane, − Lane: PCR product from untreated sample. Yellow arrows serve to highlight differential methylation between matched control and tumor samples for a given patient, where a visible band underneath the PCR product indicates the presence of 5mC and no visible band indicates no 5mC presence. (b,c) 5-hydroxymethylcytosine (5hmC) abundance in SPRY2 gene bodies in colon cancers and matched adjacent mucosa (n = 12 samples, p < 0.05, paired Student’s t-test): (b) Increase in mean log2 fold change of 5hmC in SPRY2 gene promoter and a marginal increase in the gene body in colon tumors (Tumor) compared to normal colon (Adjacent). (c) 5hmC peaks within the gene bodies of SPRY2 in tumor and normal colon. The moving averages at 0.01 smoother span are shown. Black bars mark exons.
Fig 2: (a) GEO dataset (GSE8671) displaying increased SPRY2 transcripts in 32 adenomas compared to matched adjacent control colon samples. Increased SPRY2 transcript expression in adenocarcinomas compared to adjacent control from (b) GEO dataset (GSE166427) and (c) The Cancer Genome Atlas (TCGA). (d) Increased SPRY2 mRNA in adenocarcinomas compared to healthy mucosa (GSE166427). (e) Increased SPRY2 transcripts in microsatellite instable (MSI) CRC tumors compared to healthy mucosa (GSE24514) (f) Copy number variation score and its values representing no copy gain (0) or gain (1) of SPRY2, where no statistical difference in transcript expression (FPKM) was observed. (g) Western blot of SPRY2 showing increased expression in tumor colon (T) compared to normal colon (A) in #1-#6 CRC patients.
Fig 3: Down-regulation of SPRY2 mRNA is associated with reduced overall and disease-free survival in patients with high-grade serous ovarian carcinomaThe cBioPortal for Cancer Genomics was used to query high-grade serous ovarian carcinomas from The Cancer Genome Atlas (n=489) for patients with altered SPRY2 mRNA levels. A. OncoPrint results showing cases with altered SPRY2 mRNA levels across all 489 high-grade serous ovarian carcinomas. B. Overall (left panel) and disease-free (right panel) survival differences between samples without and with up-regulated SPRY2 mRNA were displayed as Kaplan-Meier survival curves with the associated P value (Log-rank test). C. Overall (left panel) and disease-free (right panel) survival differences between samples without and with down-regulated SPRY2 mRNA were displayed as Kaplan-Meier survival curves with the associated P value (Log-rank test).
Fig 4: AREG up-regulates SPRY2 expression through EGFR in another human ovarian cancer cell lineA. OVCAR5 cells were treated with 10 ng/mL AREG (AR) for different time periods, and SPRY2 protein levels were examined by Western blot. B. OVCAR5 cells were pre-treated with vehicle control (DMSO) or 10 μM AG1478 for 1 h and then treated with vehicle control (Ctrl) or 10 ng/mL AREG (AR) for 3 h. SPRY2 protein levels were examined by Western blot. C. OVCAR5 cells were transfected with 50 nM control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 h and then treated with vehicle control (Ctrl) or 10 ng/mL AREG (AR) for 3 h. The protein levels of SPRY2 and EGFR were examined by Western blot. The results are presented as the mean ± SEM of at least three independent experiments. Values without a common letter are significantly different (P < 0.05).
Fig 5: EGFR is required for the AREG-induced up-regulation of SPRY2 expressionA and B. SKOV3 cells were pre-treated with vehicle control (DMSO) or 10 μM AG1478 for 1 h and then treated with vehicle control (Ctrl) or 10 ng/mL AREG (AR) for 3 h. The mRNA (A) and protein (B) levels of SPRY2 were examined by RT-qPCR and Western blot, respectively. C and D. SKOV3 cells were transfected with 50 nM control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 h and then treated with vehicle control (Ctrl) or 10 ng/mL AREG (AR) for 3 h. The mRNA (A) and protein (B) levels of SPRY2 and EGFR were examined by RT-qPCR and Western blot, respectively. The results are presented as the mean ± SEM of at least three independent experiments. Values without a common letter are significantly different (P < 0.05).
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