Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: ENC1 expression is often upregulated in CRC and associated with poor clinical outcomes. (A) The ENC1 mRNA expression levels in CRC and other cancers from the Oncomine cohort. The threshold was selected with the following parameters: p-value of 1E-4, fold change of 2, and gene rank of top 10%. (B,C) Heat map and Violin plot presented the upregulated expression of ENC1 between CRC ( n = 469) and normal colon tissues ( n = 41) by bioinformatics analysis from TCGA. Unpaired t -test, ** P < 0.01. (D) ENC1 expression was overexpressed in CRC ( n = 275) compared with normal colon tissues ( n = 41) by box plot in the GEPIA database. * P < 0.05. (E) The ENC1 mRNA expression in 24 paired CRC patients quantified from qRT-PCR. Wilcoxon matched-pair test, * P < 0.05. (F) Log10-fold change was used to show the differential mRNA expression of ENC1 between CRC and normal tissues. (G) The ENC1 protein expression was detected by Western blotting in 12 pairs of CRC tissues (T) and adjacent normal tissues (NT). The scatter plot revealed the relevant density of ENC1 protein expression. (H) IHC analysis of ENC1 expression in CRC TMAs. Representative stage images were shown (upper: magnification × 40, scale bar = 500 μm; lower: magnification × 200, scale bar = 100 μm). Box plot described the IHC score of ENC1 in 100 CRC tissues (IHC score: 9.17 ± 2.719) and 78 matched peritumoral tissues (IHC score: 2.397 ± 1.59). Mean ± SD, unpaired t -test, *** P < 0.001. (I) Kaplan–Meier’s overall survival curve showed that the patients with a higher expression level of ENC1 have a shorter survival period. (J) Univariate and (K) multivariate Cox proportional hazard analyses were conducted to evaluate the HR of ENC1 for overall survival in CRC.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: Correlation between ENC1 expression and clinicopathological features in 100 CRC patients.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: ENC1 regulates proliferation, migration, and invasion of CRC cells. (A) Endogenous ENC1 protein expression status was tested in human normal colonic epithelial cells (NCM460) and CRC cells (HT29, LOVO, DLD-1, SW620, HCT116, SW480, and RKO) by Western blotting. (B) Endogenous ENC1 mRNA expression status was compared between NCM460 and CRC cells by qRT-PCR. Western blot analysis of ENC1 protein expression levels in ENC1 overexpression (C) and ENC1 knockdown (D) CRC cells HT29/HCT116. (E,F) CCK-8 and (G,H) colony formation assays were performed to assess cell proliferation in CRC cells with ENC1 overexpression or knockdown. Wound-healing assay was used to detect cell migration ability in ENC1 overexpression (I) and ENC1 knockdown (J) CRC cells. Transwell assays showed the effect of ENC1 overexpression (K) and ENC1 knockdown (L) on CRC cell migration and invasion (left: representative images; right: quantitative analyses). Results are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Migration, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, CCK-8 Assay, Wound Healing Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: ENC1 overexpression induces EMT and maintains stemness of CRC. (A,B) The ENC1 overexpression or knockdown efficiency was assessed in ENC1-overexpression and ENC1-knockdown HT29/HCT116 cells by qRT-PCR. (C,D) Immunofluorescence staining of E-cadherin and Vimentin expression in overexpressed or silenced HT29 (magnification × 20, scale bar = 50 μm), and the cell nuclei were stained with DAPI in blue. (E,F) EMT markers of E-cadherin, N-cadherin, Vimentin, and Snail protein expression levels in ENC1-overexpression and ENC1-knockdown CRC cells were detected by Western blotting. Representative images of sphere formation and sphere number analysis were displayed (magnification × 40, scale bar = 200 μm) in HT29/HCT116 after ENC1 overexpression (G) and ENC1 knockdown (H) . (I) Flow cytometry analysis of CD44-positive cells in the indicated groups of cells. (J,K) Western blotting analysis of stemness markers (CD44, CD133, and SOX2) expression in ENC1-overexpression and ENC1-knockdown CRC cells. Results are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Over Expression, Knockdown, Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Western Blot, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: In vivo , downregulation of ENC1 inhibits xenograft tumor growth and lung metastasis. (A) Images of xenograft tumors from six BALB/c nude mice at 42 days after subcutaneously inoculated with HCT116-Ctrl shRNA and HCT116-ENC1 shRNA-1 cells. (B) The growth of xenograft tumors was measured by tumor volume every 4 days and tumor growth curves were plotted between the comparison in two groups ( n = 6). (C) Representative images of IHC and H&E staining show that ENC1 silencing downregulated the expression of ENC1 and Ki-67 in xenograft tumors (magnification × 400, scale bar = 50 μm). The protein and mRNA levels of ENC1 were also analyzed in xenograft tumor tissues by Western blotting (D) and qRT-PCR (E) , respectively. (F) Representative images of metastatic lungs in the HCT116-Ctrl shRNA (left) group and HCT116-ENC1 shRNA-1 (right) group. Black arrows indicated the metastatic pulmonary nodules. (G) H&E staining (upper panel: magnification × 100, scale bar = 200 μm) and IHC staining with ENC1 (lower panel: magnification × 400, scale bar = 50 μm) were performed on section of metastatic pulmonary nodules. Results are presented as the mean ± SD of three independent experiments. Student’s t test was applied for statistical analysis in two-group comparison: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: In Vivo, shRNA, Comparison, Staining, Expressing, Western Blot, Quantitative RT-PCR, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: ENC1 activates JAK2/STAT5/AKT signaling in CRC. (A) Top five significantly and positively enriched pathways with high ENC1 expression by GSEA. (B) ENC1 expression positively correlated with STAT5-activated gene signatures (PID_IL2_STAT5_PATHWAY). (C,D) Western blotting analysis of JAK2/STAT5 signaling key members (p-JAK2, p-STAT5, p-AKT, JAK2, STAT5, and AKT) in HT29/HCT116 after ENC1 overexpression and ENC1 knockdown. (E) ENC1-overexpressed HT29/HCT116 were treated with JAK1/2 inhibitor ruxolitinib. After being treated for 24 h, as indicated, p-JAK2, p-STAT5, p-AKT, JAK2, STAT5, and AKT expression levels were detected by western blotting. Results are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Expressing, Western Blot, Over Expression, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: ENC1 Facilitates Colorectal Carcinoma Tumorigenesis and Metastasis via JAK2/STAT5/AKT Axis-Mediated Epithelial Mesenchymal Transition and Stemness

doi: 10.3389/fcell.2021.616887

Figure Lengend Snippet: ENC1 accelerates cell proliferation, migration, invasion, EMT, and stemness through activating JAK2/STAT5/AKT signaling in CRC. Cell proliferation was assessed by CCK8 assay (A) and colony formation assay (B) in ENC1-overexpression CRC cells treated with ruxolitinib. (C) Effects of ruxolitinib treatment on ENC1-overexpressed HT29/HCT116 cell motility and invasiveness capability were detected by Transwell assay. (D) Western blotting showed the protein changes of EMT markers between ENC1-overexpressed cells with and without ruxolitinib treatment. (E) Effect of ruxolitinib in tumor spheres caused by ENC1 overexpression in contrast with no treatment. (F) The expression of stemness markers (CD44, CD133, SOX2) were detected by Western blotting in ENC1-overexpressed CRC cells treated with ruxolitinib. (G) Schematic diagram of ENC1 promotes CRC tumorigenesis and metastasis via the JAK2/STAT5/AKT axis mediated EMT and stemness. Results are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

Techniques: Migration, CCK-8 Assay, Colony Assay, Over Expression, Transwell Assay, Western Blot, Expressing

Journal: Journal of Clinical Investigation

Article Title: CCL28-induced RARβ expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/jci125336

Figure Lengend Snippet: Figure 8. Expression levels of CCL28, CCR10, or RARβ are closely associated with overall survival in 117 patients with OSCC. (A) Representative images of IHC staining of CCL28, CCR3, CCR10, and RARβ in normal oral mucosa and OSCC tissues. Scale bars: 100 μm. Magnified images of the boxed area are shown in the insets. Scale bars: 20 μm. (B) Frequency of histoscores in normal oral mucosa and OSCC tissues. (C) Kaplan-Meier survival curve of patients with OSCC stratified based on CCL28, CCR3, CCR10, or RARβ expres- sion by the log-rank test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: High expression of ENC1 in lung cancer tissues and cells. (A and B) The expression of ENC1 between lung cancer and para-cancerous tissue from the TCGA database. (C) Kaplan-Meier survival plot of overall survival of lung cancer in TCGA database, categorized according to ENC1 gene expression (high vs. low, based on mean expression). (D) The expression of ENC1 in normal and tumor tissues by RT-qPCR. (E) The expression of ENC1 in tumor tissues between stage II and III by RT-qPCR. (F) The expression of ENC1 in normal and tumor tissues by immunohistochemical analysis. (G and H) The expression of ENC1 between normal cell line and lung cancer cell lines by RT-qPCR and western blot analysis. Scale bar, 50 µ m; * P<0.05, *** P<0.001 vs. normal group; **P<0.01 vs. 16HBE cell group; ENC1, ectodermal-neural cortex 1.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Immunohistochemical staining, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Associations between ENC1 expression and the clinicopathological parameters of patients with lung adenocarcinoma.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Gene expression profiling of si-ENC1-transfected A549. (A) Volcano plot of all genes detected in transcriptome analysis. (B) Heatmap represents the magnitude of gene expression of 1404 genes (P<0.05, LogFC ≥1 o r≤−1). (C and D) Relative fold change of genes representing key pathways identified by GO and KEGG analyses. (E and F) Heatmap represents magnitude of gene expression of cell adhesion and regulation of cell binding. ENC1, ectodermal-neural cortex 1.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Gene Expression, Transfection, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Downregulation of ENC1 inhibits the migration and invasion of NSCLC cells. (A) Knockdown efficiency of ENC1 was quantified in A549 and H1299 by RT-qPCR. (B) Knockdown efficiency of ENC1 was quantified in A549 and H1299 by western blot analysis. (C-J) The effects of ENC1 deficiency on cell migration and invasion were investigated by Transwell assay. (K-M) The effects of ENC1 deficiency on the expression of N-cadherin, E-cadherin, MMP2 and MMP9 were investigated by western blot analysis. * P<0.05, ** P<0.01, *** P<0.001 vs. si-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Migration, Knockdown, Quantitative RT-PCR, Western Blot, Transwell Assay, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Downregulation of ENC1 inhibits the proliferation of NSCLC cells. (A and B) Effects of ENC1 deficiency on the proliferation were investigated in A549 and H1299 by CCK-8 assay. (C-F) The effects of ENC1 deficiency on the proliferation were investigated in A549 and H1299 using an EdU detection kit. * P<0.05, ** P<0.01 vs. si-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: CCK-8 Assay

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Downregulation of ENC1 affects the JNK and ERK pathways. (A-C) Effects of ENC1 deficiency on the expression of p-ERK, ERK, p-JNK, JNK p-p38 and p38 were quantified in A549 and H1299 cells by western blot analysis. * P<0.05 vs. si-NC group. ENC1, ectodermal-neural cortex 1.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Downregulation of ENC1 inhibits the growth of NSCLC in a mouse model. (A) Image of murine tissues from the 2 groups. (B) Tumor volume in the sh-ctrl and sh-ENC1 groups. (C) Tumor weight in the sh-ctrl and sh-ENC1 groups. (D) Tissue pathological changes in the sh-ctrl and sh-ENC1 groups, determined by H&E staining. (E-H) The expression levels of N-cadherin, E-cadherin, MMP2 and MMP9 were investigated and compared between the sh-ctrl and sh-ENC1 groups by immunohistochemistry. Scale bar, 50 µ m; ** P<0.01, vs. sh-ctrl. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Staining, Expressing, Immunohistochemistry

Journal: International Journal of Molecular Medicine

Article Title: Ectodermal-neural cortex 1 affects the biological function of lung cancer through the MAPK pathway

doi: 10.3892/ijmm.2021.4912

Figure Lengend Snippet: Upregulation of ENC1 enhances the proliferation, migration and invasion of NSCLC cells. (A) The overexpression efficiency of ENC1 was quantified in A549 and H1299 by RT-qPCR. (B) The overexpression efficiency of ENC1 was quantified in A549 and H1299 by western blot analysis. (C) Effects of ENC1 overexpression on proliferation were investigated in A549 and H1299 cells by CCK-8 assay. (D) The effects of ENC1 overexpression on the proliferation were investigated in A549 and H1299 cells using an EdU detection kit. (E and F) The effects of ENC1 overexpression on cell migration and invasion were investigated by Transwell assay. * P<0.05, ** P<0.01, *** P<0.001 vs. OE-NC group. ENC1, ectodermal-neural cortex 1; NSCLC, non-small cell lung cancer.

Article Snippet: For transfection, cells at approximately 60% confluency were transfected with 8 nmol/l siRNAs targeting ENC1 or ENC1 overexpression vector using the GenMute Transfection kit according to the manufacturer's instructions (SignaGen Laboratories) for 48 h. Following 12-24 h of transfection, the culture medium was replaced with fresh medium for an additional 48 h of incubation at 37°C in a humidified atmosphere with 5% CO 2 . siRNA against ENC1 (si-ENC1), non-specific siRNA (si-NC), ENC1 overexpression vector and vector were designed and synthesized by Guangzhou RiboBio Co., Ltd.

Techniques: Migration, Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transwell Assay