scramble shrna knockdown vectors  (Addgene inc)

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: PAX6-induced cancer stemness properties via SOX2. ( A ) Re-expression of the PAX6 gene after treatment of LUAD cells with 5-Aza-dC ± Trichostatin A (TSA), as determined by Q-RT-PCR. The relative expression levels were calculated as a ratio of the values of 5-Aza-dC ± TSA treatment relative to the value of mock treated cells considered as 1.0. Plus (+) and minus (-) marks represent cell lines with and without methylation of the PAX6 gene, respectively. ( B ) The PM values of the PAX6 gene in isogeneic parental and spheroid LUAD cells, as measured by Q-MSP. ( C ) Western blotting analysis of PAX6 in isogeneic parental and spheroid cells. ( D ) Sphere formation and self-renewal assays through the second (P2) passage from the first passage (P1) in stable PAX6 knockdown (PAX6-sh) NCI-H1650 and overexpressed (PAX6-LV) NCI-H1975 cells compared with control (PAX6-Ctrl) cells. Left, representative images of sphere formation (scale bars, 200 μm); Right, number of the spheres over 100 μm. ( E ) Western blotting analysis of PAX6 and SOX2 expression in stable PAX6-sh NCI-H1650 and PAX6-LV NCI-H1975 cells. ( F ) The in vivo tumorigenesis after xenotransplantation of stable PAX6-sh NCI-H1650 (left) and PAX6-LV NCI-H1975 (right) cells (four mice per group). ( G ) Limiting dilution xenograft assays in stable PAX6-sh NCI-H1650 spheroid (upper) and PAX6-LV NCI-H1975 (lower) cells. Tumor-initiating capacity is shown as the numbers of tumors / the number of injections. ( H ) Sphere formation assay (upper left) and western blotting analysis (lower left) in PAX6-sh or PAX6-Ctrl NCI-H1650 cells transduced with SOX2-LV or SOX2-Ctrl (PAX6-Ctrl/SOX2-Ctrl, PAX6-sh/SOX2-Ctrl, and PAX6-sh/SOX2-LV). Right, representative images of sphere formation (scale bars, 200 μm). The spheres over 100 μm were counted. ( I ) Sphere formation assay (upper left) and western blotting analysis (lower left) of PAX6-LV or PAX6-Ctrl NCI-H1975 cells transduced with SOX2-sh or SOX2-Ctrl (PAX6-Ctrl/SOX2-Ctrl, PAX6-LV/SOX2-Ctrl, and PAX6-LV/SOX2-sh). Right, representative images of sphere formation (scale bars, 200 μm). Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ D and F ] Chi-squared test [ G ], and Kruskal–Wallis with post-hoc test [ H and I ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, Western Blot, In Vivo, Tube Formation Assay, Transduction, MANN-WHITNEY

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: The PAX6-GLI-SOX2 signaling axis in LUAD CSCs. ( A ) Gene set enrichment analysis (GSEA) related to the oncogenic signatures and hallmarks in the groups with high and low expression of PAX6 in the TCGA LUAD cohort. Left, the enhanced oncogenic pathways in group with high PAX6 expression, as determined by a normalized enrichment score (NES); Right, the enrichment of Hedgehog pathway in group with high PAX6 expression. The GSEAs from both oncogenic signatures gene set (left and middle panels) and hallmarks gene set (right panel) showed the enriched Hedgehog pathway. SHH; Sonic hedgehog. ( B ) Western blotting analysis of GLI in stable PAX6-sh NCI-H1650 or PAX6-LV NCI-H1975 cells. ( C ) ChIP assays conducted on the proximal promoter region of the SOX2 gene using the indicated antibodies in NCI-H1650 or PAX6-LV NCI-H1975 cells. Histone H3 and normal IgG were used as the positive and negative control, respectively. ( D ) Sphere formation assay (upper left) and western blotting analysis of indicated molecules (lower left) of PAX6-LV NCI-H1975 cells transfected with GLI siRNA ± SOX2-LV. Right, representative images of sphere formation (scale bars, 200 μm). The spheres over 100 μm were counted. ( E ) Sphere formation assay (upper left) and western blotting analysis (lower left) of PAX6-sh NCI-H1650 cells stimulated with recombinant human Sonic Hedgehog ligand (Shh; 1μg /ml) after transfection with SOX2-sh or SOX2-Ctrl. Right, representative images of sphere formation (scale bars, 200 μm). ( F ) The in vivo tumorigenesis of stable PAX6-LV or PAX6-Ctrl NCI-H1975 cells in the presence or absence of the GLI antagonist GANT61 (50 mg/kg) treatment for 21 days (five mice per group). Each error bar indicates mean ± SEM. **, P <0.01 (Kruskal–Wallis with post-hoc test [ D, E, and F ]). See also

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Western Blot, Negative Control, Tube Formation Assay, Transfection, Recombinant, In Vivo

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: PAX6-regulated differentiation lineage factors (HOPX and NKX2-1) and stemness-related factors (SOX2 and GLI) in LUAD. ( A ) The enrichment scores of molecular subtype-related genes from GSEA in the groups with high and low expression of PAX6 in the TCGA LUAD cohort. The enrichment score of each gene was summarized in . The alveolar-related genes, especially HOPX and NKX2-1, were enriched in group with low PAX6 expression compared with high group. DASC, distal airway stem cell. ( B ) The relative expression levels of HOPX and NKX2-1 in PAX6-sh (NCI-H1650 and NCI-H1299) and PAX6-LV (NCI-H1975 and NCI-H23) cells compared with PAX6-Ctrl cells, as measured by Q-RT-PCR. ( C ) The promoter methylation levels of the PAX6 gene and expression levels of HOPX and NKX2-1 in re-differentiated cells compared with spheroid cells. When spheroid cells were cultured under standard conditions containing the fetal bovine serum (FBS), the floating spheroid cells could adhere and acquire epithelial morphology similar to parental cells. The cells showed higher expression of HOPX and NKX2-1 along with re-methylation of the PAX6 gene compared with corresponding spheroid cells, as measured by Q-RT-PCR and Q-MSP. ( D ) A linear correlation analysis of PAX6 with SOX2, GLI1, GLI2, HOPX, and NKX2-1 in 75 human lung cancer tissues. The relative mRNA expression levels were calculated as the Q-RT-PCR values of each molecules vs. β-actin (The values of PAX6, GLI1, and HOPX were multiplied by 1000 for easy tabulation). The extent of the correlation is indicated by the R coefficient. ( E ) The association between PAX6 expression and the status of histological differentiation (well/moderate vs. poor differentiation). Scatter plots show the distribution of relative mRNA expression values of PAX6 according to the differentiation status. ( F ) The association of PAX6 promoter methylation with the expression levels of HOPX and NKX2-1. Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ A, B, E, and F ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, Cell Culture, MANN-WHITNEY

Journal: Oncogene

Article Title: Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma

doi: 10.1038/s41388-018-0373-2

Figure Lengend Snippet: CSC expansion following chemotherapy abrogated by targeting the PAX6-GLI-SOX2 axis in xenograft models. ( A ) Sphere formation assay of cancer cells isolated from xenografted tumor tissues after treatment with pemetrexed (75 mg/kg, once daily, 5 days on and 2 days off) plus cisplatin (4 mg/kg, once every 7 days; Pem-Cis) chemotherapy for 14 days in PAX6-sh or PAX6-Ctrl xenograft models (NCI-H1650 and NCI-H1299 cells). Upper, representative images of sphere formation (scale bars, 200 μm); Lower, number of spheres over 100 μm. ( B ) The expression levels of GLI, PAX6, and SOX2 of xenografted tumor tissues after treatment with Pem-Cis chemotherapy for 14 days in PAX6-sh or PAX6-Ctrl NCI-H1650 xenograft models, as measured by western blotting analysis for GLI and PAX6 (left) and flow cytometry for SOX2 (right). ( C ) Sphere formation assay of cancer cells isolated from xenografted tumor tissues after treatment with Pem-Cis chemotherapy in the presence or absence of the GLI antagonist GANT61 (50 mg/kg) treatment for 14 days in PAX6-LV or PAX6-Ctrl NCI-H1975 xenograft models. ( D ) The promoter methylation levels of the PAX6 gene of xenografted tumor tissues after treatment with Pem-Cis chemotherapy for 14 days in xenograft models, as measured by Q-MSP. ( E ) The in vivo therapeutic efficacy of the combination of Pem-Cis chemotherapy for 14 days with GANT61 (50 mg/kg) for 21 days in NCI-H1650 and NCI-H1975 xenograft models (five mice per group). Left, tumor growth curve; Right, tumor weight from both flanks of five mice per group. Growth curves were calculated by comparing the tumor size before any treatment with the size at different time points of therapy. ( F ) The in vivo therapeutic efficacy of the combination of Pem-Cis chemotherapy with GANT61 (50 mg/kg) in patient derived xenograft (PDX) models (five mice per group). Each error bar indicates mean ± SEM. *, P <0.05; **, P <0.01 (Wilcoxon–Mann–Whitney test [ A and C ] and Kruskal–Wallis with post-hoc test [ E and F ]). See also .

Article Snippet: SOX2 shRNA pGFP-C-shLenti Vector (Cat # TL309173) for SOX2 knockdown (SOX2-sh) and Non-effective 29-mer scrambled shRNA pGFP-C-shLenti Vector (Cat # TR30021) for a control (SOX2-Ctrl) were purchased from Origene (Rockville, USA).

Techniques: Tube Formation Assay, Isolation, Expressing, Western Blot, Flow Cytometry, Methylation, In Vivo, Derivative Assay, MANN-WHITNEY