Figures & data
Table 1. Clinicopathological features of patients with colon adenocarcinoma
Table 2. Sequence of NOTUM lentivirus
Figure 1. Bioinformatic analysis of the role of NOTUM in COAD and other digestive system carcinomas using the GEPIA serve. (a) Specific quantified value. (b) Expression profile in different cancer types from the GEPIA databases. (c and d) Protein expression profile assay in different digestive system carcinomas. (e) Isoform analysis for NOTUM in COAD, presented as a boxplot. (f) Pathological stage plot assay for NOTUM in COAD. (g) Survival analyses in COAD cancer type. (h) Pair-wise gene correlation analysis of NOTUM and FAS. NOTUM, notum palmitoleoyl-protein carboxylesterase; COAD, colon adenocarcinoma; FAS, Fas cell surface death receptor; T, tumor; N, normal; TPM, transcripts per million
Figure 2. Expression levels of NOTUM in different tissues or cell lines. (a) Western blot analysis of NOTUM expression in tumor and para-tumor tissues, and (b) semi-quantification of results. (c) RT-qPCR assay for mRNA expression of NOTUM in tumor and para-tumor tissues. (d) Western blot analysis of NOTUM expression in NCM460, RKO, HCT116 and SW620 cells, and (e) semi-quantification of results. (f) RT-qPCR assay for mRNA expression of NOTUM in NCM460, RKO, HCT116 and SW620 cells. Protein and mRNA expression levels were normalized to β-actin. n = 6 per group. Data are presented as the mean ± SD. *P < 0.05, vs. para-tumor or NCM460 cells. NOTUM, notum palmitoleoyl-protein carboxylesterase; RT-qPCR, reverse transcription-quantitative PCR
Figure 3. Knockdown of NOTUM regulates colon adenocarcinoma development and apoptosis in vitro. (a) Proliferation assay was conducted over 5 days. (b) PI staining assay was performed to examine apoptosis. (c) PI (+) cells assay was performed in Con, sh-NOTUM and control-shNOTUM groups in vitro. (d) Western blot analysis of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels in vitro. (e) Semi-quantification of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels. Protein levels were normalized to β-actin. n = 6 per group. *P < 0.05, Con and control-shNOTUM groups vs. sh-NOTUM group. NOTUM, notum palmitoleoyl-protein carboxylesterase; FAS, Fas cell surface death receptor; PARP1, poly (ADP-ribose) polymerase 1; Con, control; shRNA, short hairpin RNA; OD, optical density
Figure 4. Knockdown of NOTUM regulates colon adenocarcinoma development and apoptosis in vivo. (a) Tumor volume over 28 days period and (b) tumor weight at 28 day in the Con, sh-NOTUM and control-shNOTUM mice. (c and d) Immunofluorescence assay of TUNEL staining (magnification, x400) and (d) TUNEL(+) cells. (e) Western blot analysis of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels in vivo. (f) Semi-quantification of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels. Protein levels were normalized to β-actin. *P < 0.05, Con and control-shNOTUM groups vs. sh-NOTUM group. (g) Survival analysis of Con, sh-NOTUM and control-shNOTUM mice. n = 6 per group. NOTUM, notum palmitoleoyl-protein carboxylesterase; FAS, Fas cell surface death receptor; PARP1, poly(ADP-ribose) polymerase 1; Con, control; shRNA, short hairpin RNA
Figure 5. NOTUM regulates FAS-mediated apoptosis via the Wnt signaling pathway (a) PI staining assay of apoptosis and (b) quantification of PI (+) cells in Con, sh-NOTUM + Vehicle and sh-NOTUM + SKL2001 mice. (c) Western blot analysis of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels in vitro. (d) Semi-quantification of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels. (e) Immunofluorescence assay of TUNEL staining (magnification, x400) and (f) number of TUNEL(+) cells in Con, sh-NOTUM + Vehicle and sh-NOTUM + SKL2001 mice. (g) Western blot analysis of NOTUM, FAS, BAX, Bcl-2, cleaved caspase-3 and cleaved PARP1 expression levels in vivo, and (h) semi-quantification of results. Protein levels were normalized to β-actin. n = 6 per group. *P < 0.05, sh-NOTUM + vehicle group vs. Con group; #P < 0.05, sh-NOTUM + SKL2001 group vs. sh-NOTUM + vehicle group. NOTUM, notum palmitoleoyl-protein carboxylesterase; FAS, Fas cell surface death receptor; PARP1, poly(ADP-ribose) polymerase 1; Con, control; shRNA, short hairpin RNA
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.