hairpin rna sequence against human sox9 shsox9  (Addgene inc)

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Analysis of side population (SP) in human lung carcinoma H460 cells in the presence or absence of fumitremorgin C (FTC) using FACS. SP cells ( box) were determined by their disappearance in the presence of FTC and were shown as percentage of the pool population. CSCs were isolated based on SP phenotype and their aggressive features were validated in vitro and in vivo as shown in . ( b ) Analysis of EMT markers and ABCG2 transporter in human normal lung epithelial BEAS-2B (BC) cells and SP (CSC) and NSP (non-CSC) H460 cells using Western blotting. Immunoblot signals from three-independent experiments (one of which is shown here) were quantified by densitometry, revealing a dominant overexpression of SLUG in SP cells. ( c ) Western blot analysis of SLUG, VIM and CDH1 in SLUG knockdown (shSNAI2) and control (shCON) H460 cells. ( d ) Protein expression of SLUG and SOX9 in clinical lung cancer and matched normal lung tissues. Blots were reprobed with anti-β-actin (ACTB) antibody to confirm equal loading of the samples. Quantitative analysis of SLUG and SOX9 levels revealed a striking difference between normal (N) and tumor (T) tissues at the significance level in two-sided Student’s t -test of P < 0.03 and P < 0.003, respectively. ( e and f ) SLUG and SOX9 knockdown and overexpression experiments were performed using H460 cells treated with lentiviral particles carrying shSNAI2, shSOX9 or shCON and nucleofection of GFP, SNAI2 or SOX9 overexpression plasmids, as described under “Materials and methods”. Analysis of ( e ) tumor sphere formation and ( f ) SP in various clones of H460 cells. Scale bar = 200 µm. Data are mean ± S.D. (n = 4). * P < 0.05 vs . shCON cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Isolation, In Vitro, In Vivo, Western Blot, Over Expression, Knockdown, Control, Expressing, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Immunohistochemistry analysis of SLUG and SOX9 in isolated lungs from mice bearing shSNAI2, shSOX9 and shCON cells. Scale bar = 200 µm. ( b ) Correlation analysis of the protein expression of SLUG and SOX9 in clinical lung tumor ( red triangle ) and matched normal ( blue square ) samples. ( c ) Western blot analysis of SLUG and SOX9 expression in shSNAI2, shSOX9 and shCon H460 and A549 cells. ( d ) shSNAI2 and shCON cell lysates were prepared, immunoprecipitated with control IgG or anti-SOX9 antibody, and probed with anti-SLUG antibody. Immunoblots were performed on cell lysates used as input for immunoprecipitation (IP) using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( e ) Chromatin IP (ChIP) analysis of SLUG binding on SOX9 promoter. Sheared chromatin was immunoprecipitated using ChIP-grade anti-SLUG antibody or IgG control, and ChIP DNA was quantified by real-time PCR with primers specific to human SOX9 promoter (see for primer sequences). Data are mean ± S.D. (n = 3). ( f ) SLUG overexpressing or knockdown H460 cells were seeded onto type I collagen-coated slides. The cells were immunostained for SLUG ( red ), SOX9 ( blue ) and examined under a confocal fluorescence microscope. Colocalization of SLUG and SOX9 is shown in the merged display ( purple ). Images were taken pairwise with the same instrumental setting. Scale bar = 50 µm. ( g ) In-cell co-IP using proximity ligation (Duolink ® ) assay as described under “Materials and methods”. Cells were similarly seeded onto type I collagen-coated slides and in-cell SLUG-SOX9 interaction ( red ) was determined using the Duolink ® assay. Scale bar = 50 µm.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Immunohistochemistry, Isolation, Expressing, Western Blot, Immunoprecipitation, Control, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Knockdown, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Ligation

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) H460 cells were treated with proteasome inhibitor MG132 (25–100 µM) for 24 hours and SOX9 expression was determined by Western blotting. ( b ) Quantitative real-time PCR of SOX9 mRNA expression in SLUG overexpressing or knockdown H460 cells. ( c ) H460 cells were treated with MG132 (50 µM) to prevent proteasomal degradation of SOX9 and cell lysates were prepared and immunoprecipitated using control IgG or anti-SOX9 antibody. The immune complexes were analyzed for ubiquitin at various times (0–3 hours) by Western blotting. Immunoblots were performed on cell lysates used as IP input using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( d ) Analysis of SOX9 ubiquitination (Poly Ub SOX9) in shSNAI2 and shCON H460 and A549 cells in the presence or absence of MG132 at 3 hours, in which ubiquitination was found to be maximal. Equal amounts of protein were loaded in each lane. Data are mean ± S.D. (n = 3). * P < 0.05 vs . shCON cells in the presence of MG132; two-sided Student’s t -test. ( e ) Various clones of H460 cells, including shCON, shSNAI2, shCON/SNAI2, shSNAI2/SNAI2, shCON/SOX9 and shSNAI2/SOX9 cells were treated with protein translation inhibitor cycloheximide (CHX; 10 µg/mL) for various times to follow the degradation of SOX9 protein. Cell lysates were prepared and SOX9 expression was determined by Western blotting. Representative immunoblots of SOX9 and loading control β-actin (ACTB) are shown (see also ). SOX9 expression time profile was plotted and SOX9 half-life was calculated and shown. Data are mean ± S.D (n = 3). * P < 0.05 vs . shCON cells; two-sided Student’s t -test. No significant differences were observed when compared between shSNAI2 and shSNAI2/SOX9 cells.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Immunoprecipitation, Control, Ubiquitin Proteomics, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP control plasmid and were analyzed for tumor sphere formation. Scale bar = 300 µm. ( b,c ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP plasmid and injected into NSG mice via tail vein at the dose of 1×10 6 cells/mouse. ( b ) Representative bioluminescence of mice taken at the time of inoculation (week 0) and at 2 and 3 weeks post-injection ( top ) and H&E micrographs of lung tissues ( bottom ). Scale bar = 200 µm. ( c ) Normalization of tumor signals at various times to their initial signal at week 0 and relative to shCON/GFP or shSNAI2/GFP cells. Data are mean ± S.D. (n = 3 or 4). * P < 0.05 vs . shCON/GFP or shSNAI2/GFP cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Labeling, Transfection, Control, Plasmid Preparation, Injection

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: In metastatic tumor cells, increased SLUG expression stabilizes SOX9 through their binding interaction, which inhibits SOX9 ubiquitination and proteasomal degradation. SOX9 stabilization promotes the expansion of CSCs and subsequent cancer metastasis.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Binding Assay, Ubiquitin Proteomics

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Analysis of side population (SP) in human lung carcinoma H460 cells in the presence or absence of fumitremorgin C (FTC) using FACS. SP cells ( box) were determined by their disappearance in the presence of FTC and were shown as percentage of the pool population. CSCs were isolated based on SP phenotype and their aggressive features were validated in vitro and in vivo as shown in . ( b ) Analysis of EMT markers and ABCG2 transporter in human normal lung epithelial BEAS-2B (BC) cells and SP (CSC) and NSP (non-CSC) H460 cells using Western blotting. Immunoblot signals from three-independent experiments (one of which is shown here) were quantified by densitometry, revealing a dominant overexpression of SLUG in SP cells. ( c ) Western blot analysis of SLUG, VIM and CDH1 in SLUG knockdown (shSNAI2) and control (shCON) H460 cells. ( d ) Protein expression of SLUG and SOX9 in clinical lung cancer and matched normal lung tissues. Blots were reprobed with anti-β-actin (ACTB) antibody to confirm equal loading of the samples. Quantitative analysis of SLUG and SOX9 levels revealed a striking difference between normal (N) and tumor (T) tissues at the significance level in two-sided Student’s t -test of P < 0.03 and P < 0.003, respectively. ( e and f ) SLUG and SOX9 knockdown and overexpression experiments were performed using H460 cells treated with lentiviral particles carrying shSNAI2, shSOX9 or shCON and nucleofection of GFP, SNAI2 or SOX9 overexpression plasmids, as described under “Materials and methods”. Analysis of ( e ) tumor sphere formation and ( f ) SP in various clones of H460 cells. Scale bar = 200 µm. Data are mean ± S.D. (n = 4). * P < 0.05 vs . shCON cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Isolation, In Vitro, In Vivo, Western Blot, Over Expression, Knockdown, Control, Expressing, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Immunohistochemistry analysis of SLUG and SOX9 in isolated lungs from mice bearing shSNAI2, shSOX9 and shCON cells. Scale bar = 200 µm. ( b ) Correlation analysis of the protein expression of SLUG and SOX9 in clinical lung tumor ( red triangle ) and matched normal ( blue square ) samples. ( c ) Western blot analysis of SLUG and SOX9 expression in shSNAI2, shSOX9 and shCon H460 and A549 cells. ( d ) shSNAI2 and shCON cell lysates were prepared, immunoprecipitated with control IgG or anti-SOX9 antibody, and probed with anti-SLUG antibody. Immunoblots were performed on cell lysates used as input for immunoprecipitation (IP) using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( e ) Chromatin IP (ChIP) analysis of SLUG binding on SOX9 promoter. Sheared chromatin was immunoprecipitated using ChIP-grade anti-SLUG antibody or IgG control, and ChIP DNA was quantified by real-time PCR with primers specific to human SOX9 promoter (see for primer sequences). Data are mean ± S.D. (n = 3). ( f ) SLUG overexpressing or knockdown H460 cells were seeded onto type I collagen-coated slides. The cells were immunostained for SLUG ( red ), SOX9 ( blue ) and examined under a confocal fluorescence microscope. Colocalization of SLUG and SOX9 is shown in the merged display ( purple ). Images were taken pairwise with the same instrumental setting. Scale bar = 50 µm. ( g ) In-cell co-IP using proximity ligation (Duolink ® ) assay as described under “Materials and methods”. Cells were similarly seeded onto type I collagen-coated slides and in-cell SLUG-SOX9 interaction ( red ) was determined using the Duolink ® assay. Scale bar = 50 µm.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Immunohistochemistry, Isolation, Expressing, Western Blot, Immunoprecipitation, Control, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Knockdown, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Ligation

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) H460 cells were treated with proteasome inhibitor MG132 (25–100 µM) for 24 hours and SOX9 expression was determined by Western blotting. ( b ) Quantitative real-time PCR of SOX9 mRNA expression in SLUG overexpressing or knockdown H460 cells. ( c ) H460 cells were treated with MG132 (50 µM) to prevent proteasomal degradation of SOX9 and cell lysates were prepared and immunoprecipitated using control IgG or anti-SOX9 antibody. The immune complexes were analyzed for ubiquitin at various times (0–3 hours) by Western blotting. Immunoblots were performed on cell lysates used as IP input using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( d ) Analysis of SOX9 ubiquitination (Poly Ub SOX9) in shSNAI2 and shCON H460 and A549 cells in the presence or absence of MG132 at 3 hours, in which ubiquitination was found to be maximal. Equal amounts of protein were loaded in each lane. Data are mean ± S.D. (n = 3). * P < 0.05 vs . shCON cells in the presence of MG132; two-sided Student’s t -test. ( e ) Various clones of H460 cells, including shCON, shSNAI2, shCON/SNAI2, shSNAI2/SNAI2, shCON/SOX9 and shSNAI2/SOX9 cells were treated with protein translation inhibitor cycloheximide (CHX; 10 µg/mL) for various times to follow the degradation of SOX9 protein. Cell lysates were prepared and SOX9 expression was determined by Western blotting. Representative immunoblots of SOX9 and loading control β-actin (ACTB) are shown (see also ). SOX9 expression time profile was plotted and SOX9 half-life was calculated and shown. Data are mean ± S.D (n = 3). * P < 0.05 vs . shCON cells; two-sided Student’s t -test. No significant differences were observed when compared between shSNAI2 and shSNAI2/SOX9 cells.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Immunoprecipitation, Control, Ubiquitin Proteomics, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP control plasmid and were analyzed for tumor sphere formation. Scale bar = 300 µm. ( b,c ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP plasmid and injected into NSG mice via tail vein at the dose of 1×10 6 cells/mouse. ( b ) Representative bioluminescence of mice taken at the time of inoculation (week 0) and at 2 and 3 weeks post-injection ( top ) and H&E micrographs of lung tissues ( bottom ). Scale bar = 200 µm. ( c ) Normalization of tumor signals at various times to their initial signal at week 0 and relative to shCON/GFP or shSNAI2/GFP cells. Data are mean ± S.D. (n = 3 or 4). * P < 0.05 vs . shCON/GFP or shSNAI2/GFP cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Labeling, Transfection, Control, Plasmid Preparation, Injection

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: In metastatic tumor cells, increased SLUG expression stabilizes SOX9 through their binding interaction, which inhibits SOX9 ubiquitination and proteasomal degradation. SOX9 stabilization promotes the expansion of CSCs and subsequent cancer metastasis.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Binding Assay, Ubiquitin Proteomics