Fig 1: SNTB1 silencing reduces expression of stemness markers, inhibits spherogenicity, and decreases SP. (A) Heatmap plot of differentially expressed cancer stem cell-related genes in shSNTB1 versus shVec. cells. Upregulated genes: depicted in red, log2 fold change >2; downregulated genes: depicted in blue, log2 fold change <–2. (B) Detection of the cancer stem cell surface markers, CD133, LGR5, and EpCAM using Western blot. Quantification of cancer stem cell surface protein levels are normalized to the a-tubulin level. (C) Representative micrographs of tumor spheres formed by the indicated SW620 and CW-2 cells with shVec. or shSNTB1 transduction (200×). Histograms show the percentage of sphere formation. (D) Flow cytometry-based Hoechst 33342 efflux assay evaluates the proportion of the SP in shSNTB1 versus shVec. cells. Histograms show the percentage of SP cells. *, P<0.05. SNTB1, beta-1 syntrophin; SP, side population; LGR5, leucine-rich repeat-containing G-protein-coupled receptor 5; EpCAM, epithelial cell adhesion molecule.
Fig 2: The expression of SNTB1 is upregulated in CRC. (A) SNTB1 mRNA levels are assessed by analyzing 459 clinical CC specimens and 41 cases of ANT from TCGA cohort. SNTB1 mRNA expression is elevated in colon cancer samples (P<0.001). (B) The comparison of SNTB1 mRNA expression in 41 cases of CC and their paired ANT tissues (P<0.001). (C) SNTB1 mRNA levels are examined by analyzing 166 RC specimens and 9 cases of ANT specimens from TCGA cohort. SNTB1 mRNA expression is elevated in RC samples (P<0.001). (D) The comparison of SNTB1 mRNA expression in 9 cases of RC and paired ANT tissues (P<0.01). (E) RT-PCR analysis of the relative SNTB1 mRNA expression in 10 CRC patient specimens and their respective ANT samples. mRNA expression is normalized to that of GAPDH. (F) Western blot measurement of SNTB1 protein levels in 10 CRC patient specimens and their respective ANT samples. a-tubulin is used as the loading control. (G) Quantification of SNTB1 protein level from data in (F). The levels of SNTB1 are normalized to the a-tubulin level. (H) SNTB1 mRNA expression is determined by RT-PCR in eight CRC cell lines, human primary CECs, and RECs. (I) SNTB1 protein expression is examined by Western blot in eight CRC cell lines and human primary colonic/rectal epithelial cells. (J) Quantification of SNTB1 protein level from data in (I). The levels of SNTB1 are normalized to the a-tubulin level. *, P<0.05; **, P<0.01. SNTB1, beta-1 syntrophin; CRC, colorectal cancer; CC, colonic cancer; ANT, adjacent normal tissues; TCGA, The Cancer Genome Atlas; RC, rectal cancer; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CECs, colonic epithelial cells; RECs, rectal epithelial cells.
Fig 3: Activation of the ß-catenin pathway by SKL2001 counteracts the phenotypes induced by SNTB1 silencing. (A) CCK8 assays measure cell viability in SW620 cells with the indicated treatment. (B) CCK8 assays measure cell viability in CW-2 cells with the indicated treatment. (C) Colony number quantification in CRC cells with the indicated treatment. (D) Quantification of the relative colony number in the indicated CRC cells. (E) Percentage of sphere formed by the indicated CRC cells. (F) Percentage of SP cells within the indicated CRC cells. *, P<0.05. SP, side population; CRC, colorectal cancer.
Fig 4: SNTB1 knockdown suppresses tumor growth in a CRC xenograft model. (A) Representative images of xenografted tumors incorporating shVec. or shSNTB1 transfection. Tumors are extracted from mice on day 35 of tumor growth. (B) Tumor size is measured every week since the second week post-injection, and the tumor volume is calculated using the following formula: volume = (width)2 × length/2 (n=6/group, P<0.05). (C) The weight of each excised tumor is determined (n=6/group, P<0.05). (D) H&E staining images of the tumors collected from the xenograft nude mice. Magnification: 100× for the upper panel and 400× for the lower panel. *, P<0.05. SNTB1, beta-1 syntrophin; H&E, hematoxylin and eosin.
Fig 5: Elevated SNTB1 expression correlates with malignant clinicopathologic characteristics in CRC. (A) Representative micrographs of immunohistochemical staining of SNTB1 in non-cancerous tissues and CRC tissues at various pathological stages. Positive staining of SNTB1 is mainly found in advanced pathological stages. (B) Distribution of SNTB1 immunostaining index in CRC samples. The intensity of SNTB1 immunostaining ranges from score 0 (absent) to score 12 (intense), and the median SI is 4. P<0.05, Fisher’s exact test. (C,D,E,F) Association between SNTB1 expression signature and clinicopathologic tumor stages. Histograms displaying different SNTB1 expression observed in patient samples stratified based on T stage (C), N stage (D), metastasis classification (E), and the AJCC staging system (F). (G,H) Kaplan-Meier curves of overall survival analysis in the two sets of CRC samples with high- or low-SNTB1 expression. Sample source: clinical patient specimens (G), the TCGA cohort (H). (I,J) Kaplan-Meier profiles of progression-free survival rates in the two sets of CRC samples with high- or low-SNTB1 expression. Sample source: clinical patient specimens (I), the TCGA cohort (J). SNTB1, beta-1 syntrophin; CRC, colorectal cancer; SI, staining index; AJCC, American Joint Committee on Cancer; TCGA, The Cancer Genome Atlas.
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