Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptional Regulation of De Novo Lipogenesis by SIX1 in Liver Cancer Cells.

doi: 10.1002/advs.202404229

Figure Lengend Snippet: Figure 3. SIX1 promotes lipogenic gene expression through association with AIB1 and HBO1. A, B) qRT-PCR (A) and immunoblot (B) analysis of AIB1/HBO1 WT or KO HepG2 cells transfected with SIX1, HBO1, AIB1, or EV as indicated. C) ChIP analysis of SIX1, AIB1, and HBO1 occupancy on the promoters of lipogenic genes in HepG2 cells. Promoter regions of each gene represent the region containing the first or second SIX1 binding site shown in Figure 2B within the gene promoters analyzed. D) Re-ChIP analysis of the occupancy of SIX1 and AIB1 or HBO1 on the indicated lipogenic gene promoters in HepG2 cells. E) ChIP analysis of SIX1, HBO1, AIB1, and histone H3 or H4 acetylation (ac) occupancy on the indicated promoters of lipogenic genes in SIX1, AIB1, or HBO1 KO HepG2 cells. Data shown are mean ± SD of triplicate measurements. Experiments have been repeated 3 times with similar results. A two-sided Student’s t-test was used to compare the means of 2 groups. When more than 2 groups were compared, one-way ANOVA was performed. **p < 0.01 versus respective WT HepG2 cells transfected with empty vector (A). *p < 0.05, **p < 0.01 versus respective normal IgG (C-E).

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies including anti-ACLY (Proteintech; Cat# 67166-1-Ig), anti-ACC1 antibody (Proteintech; Cat# 21923-1-AP), anti-FASN antibody (Proteintech; Cat# 10624-2-AP), anti-SCD1 antibody (Proteintech; Cat# 28678-1-AP), anti-SIX1 antibody (Proteintech; Cat# 10709-1-AP), anti-AIB1 antibody (Santa Cruz Biotechnology; Cat# sc-9119), anti-HBO1 antibody (Proteintech; Cat# 13751-1-AP), and anti-β-actin (Santa Cruz Biotechnology; Cat# sc-47778HRP) at room temperature for 2 h or overnight at 4 °C.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Transfection, Binding Assay, Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptional Regulation of De Novo Lipogenesis by SIX1 in Liver Cancer Cells.

doi: 10.1002/advs.202404229

Figure Lengend Snippet: Figure 4. SIX1 is an insulin-responsive gene and stimulates de novo lipogenesis. A) Lipid droplets (LDs) were visualized using BODIPY 493/503 in SIX1 WT or KO HepG2 cells or SIX1 KO HepG2 cells transfected with SIX1. Scale bar, 20 μm. B) Long-chain fatty acid levels were analyzed by liquid chromatography-mass spectrometry in cells from (A). C) Triglyceride and total cholesterol levels were measured in cells from (A). D-F) Lipid droplets (D), long-chain fatty acid levels (E), and triglyceride and total cholesterol levels (F) were analyzed in WT or HBO1/AIB1/SCD1 KO HepG2 cells transfected with SIX1 or EV. G) Immunoblot analysis of SIX1 WT or KO HepG2 cells treated with insulin (100 nM) or linsitinib (1.0 μM). H, I) Long-chain fatty

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies including anti-ACLY (Proteintech; Cat# 67166-1-Ig), anti-ACC1 antibody (Proteintech; Cat# 21923-1-AP), anti-FASN antibody (Proteintech; Cat# 10624-2-AP), anti-SCD1 antibody (Proteintech; Cat# 28678-1-AP), anti-SIX1 antibody (Proteintech; Cat# 10709-1-AP), anti-AIB1 antibody (Santa Cruz Biotechnology; Cat# sc-9119), anti-HBO1 antibody (Proteintech; Cat# 13751-1-AP), and anti-β-actin (Santa Cruz Biotechnology; Cat# sc-47778HRP) at room temperature for 2 h or overnight at 4 °C.

Techniques: Transfection, Liquid Chromatography, Mass Spectrometry, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Transcriptional Regulation of De Novo Lipogenesis by SIX1 in Liver Cancer Cells.

doi: 10.1002/advs.202404229

Figure Lengend Snippet: Figure 7. Clinical relevance of the DGUOK-AS1/miR-145-5p/SIX1 axis in liver cancer. A) Representative IHC of 73 liver cancer patients. SIX1 and SCD1 were assessed by IHC, and DGUOK-AS1 and miR-145-5p by FISH. Scale bar: 100 μm. The correlations among DGUOK-AS1, miR-145-5p, SCD1, and SIX1 were analyzed as indicated. Case 1 and case 2 refer to 2 representative samples categorized by low and high expression of SIX1. Data was analyzed by Spearman’s Rank Correlation test. B) DGUOK-AS1 expression in 73 cancerous liver tissues and matched adjacent normal liver tissues was determined by FISH. The DGUOK-AS1 expression levels were plotted and compared between normal and cancer tissues (Mann-Whitney U test). C) The disease-free and overall survival curves related to low and high expression of DGUOK-AS1 were analyzed in 73 liver cancer patients from (A) using the Kaplan-Meier method. D) A proposed model underlying the role of the insulin/DGUOK-AS1/miR-145-5p/SIX1 axis in de novo lipogenesis and liver tumor growth and metastasis. Insulin stimulates the expression of DGUOK-AS1, thus sponging miR-145-5p. Inhibition of miR-145-5p promotes expression of SIX1, which recruits histone acetyltransferases AIB1 and HBO1 to induce expression of lipogenic genes (ACLY, FASN, and SCD1). Induction of lipogenic gene expression promotes hepatic lipogenesis and liver tumor growth and metastasis. FAs, fatty acids. TGs, triglycerides. LDs, lipid droplets.

Article Snippet: The membranes were blocked with 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies including anti-ACLY (Proteintech; Cat# 67166-1-Ig), anti-ACC1 antibody (Proteintech; Cat# 21923-1-AP), anti-FASN antibody (Proteintech; Cat# 10624-2-AP), anti-SCD1 antibody (Proteintech; Cat# 28678-1-AP), anti-SIX1 antibody (Proteintech; Cat# 10709-1-AP), anti-AIB1 antibody (Santa Cruz Biotechnology; Cat# sc-9119), anti-HBO1 antibody (Proteintech; Cat# 13751-1-AP), and anti-β-actin (Santa Cruz Biotechnology; Cat# sc-47778HRP) at room temperature for 2 h or overnight at 4 °C.

Techniques: Expressing, MANN-WHITNEY, Inhibition, Gene Expression

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: ( A ) The experimental paradigm. The differential responses to estradiol (E2) treatment of MCF-7 (cell growth) and long-term estrogen deprived MCF-7:5C cells (apoptosis) are indicated. Proteomics profiles of the two cell lines at baseline and after a brief (2 h) E2 treatment were generated using immunoprecipitations (IP). Proteins interacting with AIB1 or phosphotyrosine containing protein complexes were isolated by IP followed by mass spectrometry. Data were then subjected to an integrated bioinformatics analysis of signaling pathways and protein networks. ( B,C ) Reversal of E2-dependent effects on MCF-7 and MCF-7:5C after depletion of endogenous AIB1 protein using two different lentiviral shRNAs. MCF-7 or MCF-7:5C cells were infected with lentiviral particles expressing control or AIB1-targeting shRNAs. (B) RNAi-mediated knockdown was assayed by Western blot analysis for AIB1 relative to an actin loading control. (C) Cell growth was assayed 6 days after plating without or with E2. The E2 effect is shown relative to the respective untreated controls (mean ±S.E.M.). Closed circles: control shRNA; Open circles (red): AIB1 shRNA. #, p<0.05 E2 treatment effect vs. no treatment in control shRNA cells; *, p<0.05 E2 treatment effect in control shRNA cells vs. E2 treatment in AIB1 depleted cells. Representative data from one of at least three independent experiments are shown.

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques: Generated, Isolation, Mass Spectrometry, Infection, Expressing, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: Venn diagrams of proteins identified from anti-AIB1 ( A,C ) or anti-pY IP ( B,D ) experimental groups. ( C,D ) Proteins in combined AIB1-IP or pY-IP data sets. Individual proteins and subgroups are shown in & .

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: The thick grey line in the middle provides an arbitrary boundary between the pathways. Anti-AIB1 immunoprecipitated (AIB1-IPed) and anti-pY-immunoprecipitated proteins (pY-IPed) are indicated by red or green circles respectively (keys at the bottom). The blue circled proteins are AIB1-IPed proteins from MCF-7 (CALM1) or MCF-7:5C cells (β-catenin) under both E2-treated and untreated conditions; the purple circled one (ITPR3) is an AIB1-IPed protein from both cells only under E2 treated condition, while the yellow circled one (TYK2) is an AIB1-IPed protein from both cells under both E2 treated and untreated conditions. Proteins circled in grey are from known canonical pathways (e.g. ERK in cell growth or BAD in apoptosis) but not identified here. Solid line arrows indicate direct interactions (e.g. CDK1 phosphorylates Rap1GAP) or translocations (e.g. catenins) of proteins, while dashed arrows indicate indirect actions of proteins (e.g. AKT activate MEK through several steps). Hammer-ended lines indicate inhibitory effects on the target. Detailled pathways are shown in , , , .

Article Snippet: Western blot analyses were done as previously described , using a monoclonal antibody for AIB1 (SRC3; clone 5E11, Cell Signaling).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: ( A ) The experimental paradigm. The differential responses to estradiol (E2) treatment of MCF-7 (cell growth) and long-term estrogen deprived MCF-7:5C cells (apoptosis) are indicated. Proteomics profiles of the two cell lines at baseline and after a brief (2 h) E2 treatment were generated using immunoprecipitations (IP). Proteins interacting with AIB1 or phosphotyrosine containing protein complexes were isolated by IP followed by mass spectrometry. Data were then subjected to an integrated bioinformatics analysis of signaling pathways and protein networks. ( B,C ) Reversal of E2-dependent effects on MCF-7 and MCF-7:5C after depletion of endogenous AIB1 protein using two different lentiviral shRNAs. MCF-7 or MCF-7:5C cells were infected with lentiviral particles expressing control or AIB1-targeting shRNAs. (B) RNAi-mediated knockdown was assayed by Western blot analysis for AIB1 relative to an actin loading control. (C) Cell growth was assayed 6 days after plating without or with E2. The E2 effect is shown relative to the respective untreated controls (mean ±S.E.M.). Closed circles: control shRNA; Open circles (red): AIB1 shRNA. #, p<0.05 E2 treatment effect vs. no treatment in control shRNA cells; *, p<0.05 E2 treatment effect in control shRNA cells vs. E2 treatment in AIB1 depleted cells. Representative data from one of at least three independent experiments are shown.

Article Snippet: For the mass spectrometry analysis, protein lysates from cells treated for 2 hours with E2 or vehicle were subjected to immunoprecipitation using gamma-bind G-Sepharose beads and an anti-AIB1 monoclonal antibody (BD Biosciences) as described or an anti-phosphotyrosine monoclonal antibody (4G-10, Millipore).

Techniques: Generated, Isolation, Mass Spectrometry, Infection, Expressing, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: Venn diagrams of proteins identified from anti-AIB1 ( A,C ) or anti-pY IP ( B,D ) experimental groups. ( C,D ) Proteins in combined AIB1-IP or pY-IP data sets. Individual proteins and subgroups are shown in & .

Article Snippet: For the mass spectrometry analysis, protein lysates from cells treated for 2 hours with E2 or vehicle were subjected to immunoprecipitation using gamma-bind G-Sepharose beads and an anti-AIB1 monoclonal antibody (BD Biosciences) as described or an anti-phosphotyrosine monoclonal antibody (4G-10, Millipore).

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: The thick grey line in the middle provides an arbitrary boundary between the pathways. Anti-AIB1 immunoprecipitated (AIB1-IPed) and anti-pY-immunoprecipitated proteins (pY-IPed) are indicated by red or green circles respectively (keys at the bottom). The blue circled proteins are AIB1-IPed proteins from MCF-7 (CALM1) or MCF-7:5C cells (β-catenin) under both E2-treated and untreated conditions; the purple circled one (ITPR3) is an AIB1-IPed protein from both cells only under E2 treated condition, while the yellow circled one (TYK2) is an AIB1-IPed protein from both cells under both E2 treated and untreated conditions. Proteins circled in grey are from known canonical pathways (e.g. ERK in cell growth or BAD in apoptosis) but not identified here. Solid line arrows indicate direct interactions (e.g. CDK1 phosphorylates Rap1GAP) or translocations (e.g. catenins) of proteins, while dashed arrows indicate indirect actions of proteins (e.g. AKT activate MEK through several steps). Hammer-ended lines indicate inhibitory effects on the target. Detailled pathways are shown in , , , .

Article Snippet: For the mass spectrometry analysis, protein lysates from cells treated for 2 hours with E2 or vehicle were subjected to immunoprecipitation using gamma-bind G-Sepharose beads and an anti-AIB1 monoclonal antibody (BD Biosciences) as described or an anti-phosphotyrosine monoclonal antibody (4G-10, Millipore).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: ( A ) The experimental paradigm. The differential responses to estradiol (E2) treatment of MCF-7 (cell growth) and long-term estrogen deprived MCF-7:5C cells (apoptosis) are indicated. Proteomics profiles of the two cell lines at baseline and after a brief (2 h) E2 treatment were generated using immunoprecipitations (IP). Proteins interacting with AIB1 or phosphotyrosine containing protein complexes were isolated by IP followed by mass spectrometry. Data were then subjected to an integrated bioinformatics analysis of signaling pathways and protein networks. ( B,C ) Reversal of E2-dependent effects on MCF-7 and MCF-7:5C after depletion of endogenous AIB1 protein using two different lentiviral shRNAs. MCF-7 or MCF-7:5C cells were infected with lentiviral particles expressing control or AIB1-targeting shRNAs. (B) RNAi-mediated knockdown was assayed by Western blot analysis for AIB1 relative to an actin loading control. (C) Cell growth was assayed 6 days after plating without or with E2. The E2 effect is shown relative to the respective untreated controls (mean ±S.E.M.). Closed circles: control shRNA; Open circles (red): AIB1 shRNA. #, p<0.05 E2 treatment effect vs. no treatment in control shRNA cells; *, p<0.05 E2 treatment effect in control shRNA cells vs. E2 treatment in AIB1 depleted cells. Representative data from one of at least three independent experiments are shown.

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques: Generated, Isolation, Mass Spectrometry, Infection, Expressing, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: Venn diagrams of proteins identified from anti-AIB1 ( A,C ) or anti-pY IP ( B,D ) experimental groups. ( C,D ) Proteins in combined AIB1-IP or pY-IP data sets. Individual proteins and subgroups are shown in & .

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: The thick grey line in the middle provides an arbitrary boundary between the pathways. Anti-AIB1 immunoprecipitated (AIB1-IPed) and anti-pY-immunoprecipitated proteins (pY-IPed) are indicated by red or green circles respectively (keys at the bottom). The blue circled proteins are AIB1-IPed proteins from MCF-7 (CALM1) or MCF-7:5C cells (β-catenin) under both E2-treated and untreated conditions; the purple circled one (ITPR3) is an AIB1-IPed protein from both cells only under E2 treated condition, while the yellow circled one (TYK2) is an AIB1-IPed protein from both cells under both E2 treated and untreated conditions. Proteins circled in grey are from known canonical pathways (e.g. ERK in cell growth or BAD in apoptosis) but not identified here. Solid line arrows indicate direct interactions (e.g. CDK1 phosphorylates Rap1GAP) or translocations (e.g. catenins) of proteins, while dashed arrows indicate indirect actions of proteins (e.g. AKT activate MEK through several steps). Hammer-ended lines indicate inhibitory effects on the target. Detailled pathways are shown in , , , .

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: ( A ) The experimental paradigm. The differential responses to estradiol (E2) treatment of MCF-7 (cell growth) and long-term estrogen deprived MCF-7:5C cells (apoptosis) are indicated. Proteomics profiles of the two cell lines at baseline and after a brief (2 h) E2 treatment were generated using immunoprecipitations (IP). Proteins interacting with AIB1 or phosphotyrosine containing protein complexes were isolated by IP followed by mass spectrometry. Data were then subjected to an integrated bioinformatics analysis of signaling pathways and protein networks. ( B,C ) Reversal of E2-dependent effects on MCF-7 and MCF-7:5C after depletion of endogenous AIB1 protein using two different lentiviral shRNAs. MCF-7 or MCF-7:5C cells were infected with lentiviral particles expressing control or AIB1-targeting shRNAs. (B) RNAi-mediated knockdown was assayed by Western blot analysis for AIB1 relative to an actin loading control. (C) Cell growth was assayed 6 days after plating without or with E2. The E2 effect is shown relative to the respective untreated controls (mean ±S.E.M.). Closed circles: control shRNA; Open circles (red): AIB1 shRNA. #, p<0.05 E2 treatment effect vs. no treatment in control shRNA cells; *, p<0.05 E2 treatment effect in control shRNA cells vs. E2 treatment in AIB1 depleted cells. Representative data from one of at least three independent experiments are shown.

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques: Generated, Isolation, Mass Spectrometry, Infection, Expressing, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: Venn diagrams of proteins identified from anti-AIB1 ( A,C ) or anti-pY IP ( B,D ) experimental groups. ( C,D ) Proteins in combined AIB1-IP or pY-IP data sets. Individual proteins and subgroups are shown in & .

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques:

Journal: PLoS ONE

Article Title: Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

doi: 10.1371/journal.pone.0020410

Figure Lengend Snippet: The thick grey line in the middle provides an arbitrary boundary between the pathways. Anti-AIB1 immunoprecipitated (AIB1-IPed) and anti-pY-immunoprecipitated proteins (pY-IPed) are indicated by red or green circles respectively (keys at the bottom). The blue circled proteins are AIB1-IPed proteins from MCF-7 (CALM1) or MCF-7:5C cells (β-catenin) under both E2-treated and untreated conditions; the purple circled one (ITPR3) is an AIB1-IPed protein from both cells only under E2 treated condition, while the yellow circled one (TYK2) is an AIB1-IPed protein from both cells under both E2 treated and untreated conditions. Proteins circled in grey are from known canonical pathways (e.g. ERK in cell growth or BAD in apoptosis) but not identified here. Solid line arrows indicate direct interactions (e.g. CDK1 phosphorylates Rap1GAP) or translocations (e.g. catenins) of proteins, while dashed arrows indicate indirect actions of proteins (e.g. AKT activate MEK through several steps). Hammer-ended lines indicate inhibitory effects on the target. Detailled pathways are shown in , , , .

Article Snippet: The AIB1(1) shRNA was derived from an siRNA for AIB1 previously described , and the AIB1(2) shRNA was from Sigma (TRCN0000019703).

Techniques: Immunoprecipitation

Journal: PLoS ONE

Article Title: Downregulation of Steroid Receptor Coactivator-2 Modulates Estrogen-Responsive Genes and Stimulates Proliferation of MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0070096

Figure Lengend Snippet: (A). Quantification of SRC-2 mRNA expression in shRNA lentivirus-infected MCF-7 cells. mRNA levels of SRC-2 in a MCF-7 cell line infected with shRNA targeting SRC-2 (SRC-2 shRNA) were compared to the expression in a control shRNA MCF-7 cell line (Ctr shRNA), and in a MCF-7 cell line transduced with shRNA lentivirus targeting SRC-3 (SRC-3 shRNA). The mRNA expression of SRC-2 is relative to TBP mRNA. The results are representative of at least three independent experiments. (B). Western blotting analyses of SRC-2-depleted MCF-7 cells. MCF-7 cells infected with shRNA lentivirus targeting SRC-2 (SRC-2 shRNA) or a negative control shRNA empty vector (Ctr shRNA), were grown in phenol red-free DMEM supplemented with charcoaled stripped FBS (5%) and 17β-estradiol (10 nM) for two days. The Ctr shRNA cells were then treated with either Vehicle or 8-CPT-cAMP (150 µM), IBMX (50 µM) and forskolin (10 µM) (cAMP) for 24 hours. Immunoblotting was performed with anti-TIF2 antibody and anti-GAPDH antibody. The results shown are representative of at least three independent experiments. (C). Microarray analyses of five RNA samples isolated from five individual cell samples of shRNA control MCF-7 cells (Ctr shRNA), SRC-2 KD MCF-7 cells (SRC-2 shRNA) and control shRNA cells treated with cAMP elevating agents, as described in A. A Venn diagram shows the number of individual and overlapping sets of genes differentially expressed after SRC-2 KD (SRC-2 shRNA) and after treatment with cAMP elevating agents. To examine which genes were similarly differentially expressed between the two treated groups when compared to control, a SAM analysis with overlapping genes was performed. The fold change cut-off value ≥1.5, and q-value = 0, was used to determine differentially expressed genes.

Article Snippet: Five individual lentiviral pLKO.1-puro short hairpin (sh) RNA plasmids targeting different sequences on SRC-2/GRIP1 or SRC-3/AIB1 mRNA and a pLKO.1-puro empty vector control were purchased from Sigma Mission® RNAi (Sigma).

Techniques: Expressing, shRNA, Infection, Transduction, Western Blot, Negative Control, Plasmid Preparation, Microarray, Isolation

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Clinicopathological features of patients with UTUC

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Immunohistochemical expression of AIB1 and EIF5A2 in upper tract urothelial carcinoma tissue (×200). Notes: ( A ) AIB1 score 0 points (negative staining), ( B ) low expression of AIB1 (score 3 points), ( C ) high expression of AIB1 (score 9 points), ( D ) EIF5A2 score 0 points (negative staining), ( E ) low expression of EIF5A2 (score 4 points) and ( F ) high expression of EIF5A2 (score 12 points).

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Immunohistochemical staining, Expressing, Negative Staining

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Association of AIB1 and EIF5A2 expression with clinicopathological parameters in 109 patients with UTUC

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Kaplan–Meier plots show intravesical recurrence-free survival curves according to AIB1 expression status ( A ), low AIB1 expression (blue line), n=58; high AIB1 expression (green line), n=51. EIF5A2 expression status ( B ), low EIF5A2 expression (blue line), n=61; high EIF5A2 expression (green line), n=48.

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Kaplan–Meier survival analysis of AIB1 expression in subsets of different grades, pT and pN patients with UTUC (log-rank test). Notes: ( A ) Low-grade, probability of IVRFS of low-grade patients with UTUC: low expression (blue line), n=20; high expression (green line), n=18. ( B ) High-grade, probability of IVRFS of high-grade patients with UTUC: low expression (blue line), n=38; high expression (green line), n=33. ( C ) pTa-1, probability of IVRFS of pTa-1 patients with UTUC: low expression (blue line), n=21; high expression (green line), n=16. ( D ) pT2–4, probability of IVRFS of pT2–4 patients with UTUC: low expression (blue line), n=37; high expression (green line), n=35. ( E ) pN0, probability of IVRFS of pN0 patients with UTUC: low expression (blue line), n=53; high expression (green line), n=40. ( F ) pN1–2, probability of IVRFS of pN1–2 patients with UTUC: low expression (blue line), n=5; high expression (green line), n=11. Abbreviations: IVRFS, intravesical recurrence-free survival; UTUC, upper tract urothelial carcinoma.

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Kaplan–Meier survival analysis of postoperative intravesical chemotherapy in subsets of different expression of AIB1 and EIF5A2 (log-rank test). Notes: AIB1 (−): low AIB1 expression, AIB1(+): high AIB1 expression, EIF5A2 (−): low EIF5A2 expression, and EIF5A2 (+): high EIF5A2 expression. ( A ) AIB1(−) EIF5A2(−), probability of IVRFS of intravesical chemotherapy for patients with UTUC: no instillation (blue line), n=1; instillation (green line), n=29. ( B ) AIB1 (−) EIF5A2 (+), probability of IVRFS of intravesical chemotherapy for patients with UTUC: no instillation (blue line), n=5; instillation (green line), n=23. ( C ) AIB1(+)EIF5A2(−), probability of IVRFS of intravesical chemotherapy for patients with UTUC: no instillation (blue line), n=10; instillation (green line), n=21. ( D ) AIB1(+)EIF5A2(+), probability of IVRFS of intravesical chemotherapy for patients with UTUC: no instillation (blue line), n=6; instillation (green line), n=14. Abbreviations: IVRFS, intravesical recurrence-free survival; UTUC, upper tract urothelial carcinoma.

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Univariate and multivariate Cox regression models predicting bladder recurrence

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: Kaplan–Meier survival analysis of postoperative intravesical chemotherapy in subsets of different risk groups of the prognostic model including EIF5A2, AIB1, lymph node status and RNU (log-rank test). Note: Probability of IVRFS of all cases: no instillation (blue line), n=22; instillation (green line), n=87 ( A ); low-risk, probability of IVRFS of the low-risk group: no instillation (blue line), n=9; instillation (green line), n=46 ( B ); high-risk, probability of IVRFS of high-risk group: no instillation (blue line), n=13; instillation (green line), n=41 ( C ). Abbreviations: IVRFS, intravesical recurrence-free survival; R N U, radical nephroureterectomy.

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques:

Journal: Cancer Management and Research

Article Title: Prognostic value of AIB1 and EIF5A2 in intravesical recurrence after surgery for upper tract urothelial carcinoma

doi: 10.2147/CMAR.S185392

Figure Lengend Snippet: ROC curves comparing the predictive accuracy by the combined AIB1, EIF5A2, pN, and the surgical approach of RNU, the AIB1 alone model, the EIF5A2 alone model, the pN model, and RNU alone model for intravesical recurrence-free survival. Abbreviations: RNU, radical nephroureterectomy; ROC, receiver operating characteristic.

Article Snippet: The slides were incubated with the anti-AIB1 antibody (BD Transduction Laboratories; Lexington, KY, USA; diluted 1:100 in PBS) or anti-EIF5A2 antibody (Abnova, Jhongli, Taiwan; diluted 1:100 in PBS) in a moist chamber.

Techniques:

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Clinicopathologic correlation of AIB1 expression in cervical cancer.

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Kaplan-Meier survival analysis of the AIB1 expression in total cohort and different subsets of cervical cancer patients. A , Total cohort (high expression =57cases, low expression=51 cases). B , M0 subset (high =38 cases, low = 43 cases). C , M1 subset (high =19 cases, low = 8 cases). D , N0 subset (high =32 cases, low = 39 cases). E , non-CR subset (high =39 cases, low = 23 cases). F , III+IV subset (high = 49 cases, low = 27 cases). The P value was calculated using a log-rank test.

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Multivariate Cox regression analysis.

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Expression of AIB1 in cervical cancer. A , The levels of AIB1 proteins in three cervical cancer cell lines and non-neoplastic cervical benign tissues examined by western blot. B , Normal cervical epithelial tissues (case 30) showed normal expression of AIB1 protein with a negative staining of AIB1 in the nuclei of all cervical epithelial cells (200×). C , Cervical squamous cell carcinoma (case 17) demonstrated normal expression of AIB1, in which all tumor cells showed negative staining of AIB1 (200×). D , Low expression of AIB1 was detected in cervical squamous cell carcinoma (case 34), in which less than 10% cancer cells showed low staining of AIB1 protein in the nuclei (200×). E , High expression of AIB1 was observed in cervical squamous cell carcinoma (case 21), in which 10~70% cancer cells demonstrated positive staining of AIB1 in the nuclei (200×). F , Another cervical squamous cell carcinoma (case 36) showed high expression of AIB1, in which more than 70% cancer cells showed high staining of AIB1 protein in the nuclei (200×).

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Expressing, Western Blot, Negative Staining, Staining

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Lentivirus-mediated AIB1 silencing enhances the chemosensitivities of cervical cancer cells. A - D , Dose-response curves of cisplatin or 5-Fu in SiHa and CaSki cells. AIB1-shRNA infected SiHa and CaSki cells showed more sensitive to cisplatin and 5-Fu than parental control cells. IC50 values are shown below. Data are Mean ± SD (n=3, P<0.05). E and F , After treated cells with cisplatin (IC30) or 5-Fu (IC30) for the 24 hours, the cleaved PARP and cleaved caspase-3 were detected in AIB1-shRNA and Luc-shRNA infected SiHa and CaSki cells by western blot.

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: shRNA, Infection, Western Blot

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: Comparison of radiobiological parameters of SiHa cells infected with sh-luc or sh-AIB1.

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Infection

Journal: Journal of Cancer

Article Title: AIB1 predicts tumor response to definitive chemoradiotherapy and prognosis in cervical squamous cell carcinoma

doi: 10.7150/jca.31697

Figure Lengend Snippet: AIB1 depletion increases the sensitivity of cervical cancer cells in response to ionizing radiation. A , Clonogenic survival of SiHa-shAIB1 and SiHa-shluc cells. Cells were exposed to increasing doses of radiation as indicated. After 12 days, colonies more than 50 cells were counted and survival curves were fitted according to the linear-quadratic mode. Results were obtained from three independent experiments. All results were from three independent experiments. B , After irradiated shAIB1-Siha and shluc-Siha cells with 3Gy or 0 Gy x-ray, the cleaved caspase-3 and cleaved PARP were detected by Western blot analysis. Experiments were performed three times and a representative result is shown. C , SiHa-shAIB1 and SiHa-shluc cells were initally treated with or without IR. 36 hours later, the proportion of apoptotic cells were determined by PI/Annexin V assay. Data described are the Means ± SD of triplicates (* P <0.05, Student's t-test).

Article Snippet: Equal amounts of cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bed-ford, MA, USA) followed by incubating with primary antibodies against AIB1 (BD Transduction Laboratories); cleaved-caspase 3 (Asp175), (Cell Signaling Technology, Boston.

Techniques: Irradiation, Western Blot, Annexin V Assay