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ATCC rna interference human cervical cancer cell line hela

Functional effects of HOTAIR inhibition on <t>HeLa</t> cells. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h. HOTAIR knockdown efficiency was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. B, Apoptosis was determined by annexin V staining and flow cytometry. C, Apoptosis rate of HeLa cells at 48 h after transfection with siHOTAIR or siNC. D, Effect of HOTAIR knockdown on cell cycle progress. The percentage of cells in the G1 phase was significantly decreased whereas that in S phase and G2/M phase was increased after HOTAIR inhibition. E, HOTAIR knockdown in HeLa cells significantly inhibits cell growth. F, Effect of HOTAIR knockdown on cell invasion, as determined in a Boyden chamber assay. G, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. H, HOTAIR inhibition led to a significant reduction of cell migration as determined by a wound-healing assay. I, Quantification of the wound healing assay. Data are presented as means ± S.D. and represent results from three independent experiments. Statistically significant differences are indicated: *p < 0.05; **p < 0.01.

Rna Interference Human Cervical Cancer Cell Line Hela, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 23836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells * "

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M114.043984

Functional effects of HOTAIR inhibition on HeLa cells. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h. HOTAIR knockdown efficiency was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. B, Apoptosis was determined by annexin V staining and flow cytometry. C, Apoptosis rate of HeLa cells at 48 h after transfection with siHOTAIR or siNC. D, Effect of HOTAIR knockdown on cell cycle progress. The percentage of cells in the G1 phase was significantly decreased whereas that in S phase and G2/M phase was increased after HOTAIR inhibition. E, HOTAIR knockdown in HeLa cells significantly inhibits cell growth. F, Effect of HOTAIR knockdown on cell invasion, as determined in a Boyden chamber assay. G, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. H, HOTAIR inhibition led to a significant reduction of cell migration as determined by a wound-healing assay. I, Quantification of the wound healing assay. Data are presented as means ± S.D. and represent results from three independent experiments. Statistically significant differences are indicated: *p < 0.05; **p < 0.01.

Figure Legend Snippet: Functional effects of HOTAIR inhibition on HeLa cells. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h. HOTAIR knockdown efficiency was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. B, Apoptosis was determined by annexin V staining and flow cytometry. C, Apoptosis rate of HeLa cells at 48 h after transfection with siHOTAIR or siNC. D, Effect of HOTAIR knockdown on cell cycle progress. The percentage of cells in the G1 phase was significantly decreased whereas that in S phase and G2/M phase was increased after HOTAIR inhibition. E, HOTAIR knockdown in HeLa cells significantly inhibits cell growth. F, Effect of HOTAIR knockdown on cell invasion, as determined in a Boyden chamber assay. G, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. H, HOTAIR inhibition led to a significant reduction of cell migration as determined by a wound-healing assay. I, Quantification of the wound healing assay. Data are presented as means ± S.D. and represent results from three independent experiments. Statistically significant differences are indicated: *p < 0.05; **p < 0.01.

Techniques Used: Functional Assay, Inhibition, Transfection, Knockdown, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Boyden Chamber Assay, Migration, Wound Healing Assay

$Quantitative proteomic identification of HOTAIR-regulated proteins in HeLa cells. A, Workflow for the identification of HOTAIR-regulated proteins. HeLa cells were differentially labeled by growing them in medium containing light or heavy amino acids (SILAC). Proteins were extracted from the labeled cells 48 h after transfection with siHOTAIR or siNC, then equal amounts of protein from each sample were combined. The protein mix was separated by 12% SDS-PAGE and the resulting gel was cut into 30 sections. Each of the fractions was in-gel digested and analyzed via LC-MS/MS. B, Heatmap showing the expression of differentially expressed proteins after HOTAIR inhibition. C, PANTHER Protein Class ontology classification of the 170 proteins differentially expressed after HOTAIR expression silencing. HOTAIR-regulated proteins were classified into 22 classes.$
Figure Legend Snippet: Quantitative proteomic identification of HOTAIR-regulated proteins in HeLa cells. A, Workflow for the identification of HOTAIR-regulated proteins. HeLa cells were differentially labeled by growing them in medium containing light or heavy amino acids (SILAC). Proteins were extracted from the labeled cells 48 h after transfection with siHOTAIR or siNC, then equal amounts of protein from each sample were combined. The protein mix was separated by 12% SDS-PAGE and the resulting gel was cut into 30 sections. Each of the fractions was in-gel digested and analyzed via LC-MS/MS. B, Heatmap showing the expression of differentially expressed proteins after HOTAIR inhibition. C, PANTHER Protein Class ontology classification of the 170 proteins differentially expressed after HOTAIR expression silencing. HOTAIR-regulated proteins were classified into 22 classes.

Techniques Used: Labeling, Multiplex sample analysis, Transfection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition

VIM contributes to the effects of HOTAIR knockdown. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h and VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. B, HeLa cells were transfected with siVIM for 48 h, then VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. C, Western blot analysis of VIM protein expression 48 h after transfection with siVIM (siVIM-I, siVIM-II and siVIM-III) or siNC. GAPDH was used as an internal control. D, HeLa cells were transfected with pVIM or pEGFP. VIM expression levels were determined by Western blotting at 24 h and 48 h after transfection. EGFP serves as the negative control and GAPDH as the loading control. E, Effect of VIM knockdown and overexpression on cell invasion as determined with a wound-healing assay. F, Quantification of the wound healing assay. G, Effect of VIM knockdown and overexpression on cell invasion, as determined with a Boyden chamber assay. H, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *p < 0.05; **p < 0.01. I, HOTAIR suppressed tumor growth and regulated VIM expression in nude mice. HeLa cells transfected with siHOTAIR or siNC and HeLa cells expressing either control shRNA or shHOTAIR were injected subcutaneously into the right flank of nude mice. After 20 days, mice were sacrificed and tumors were dissected and weighed. Representative photographs of xenografts were taken 20 days after injection of HeLa cells transfected with siHOTAIR or HeLa-KD cells. J, Quantification of tumor weight. Data are presented as means ± S.D. (n = 5). K, Western blotting of VIM protein expression in tumors excised from the mice indicated 20 days after injection.

Figure Legend Snippet: VIM contributes to the effects of HOTAIR knockdown. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h and VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. B, HeLa cells were transfected with siVIM for 48 h, then VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. C, Western blot analysis of VIM protein expression 48 h after transfection with siVIM (siVIM-I, siVIM-II and siVIM-III) or siNC. GAPDH was used as an internal control. D, HeLa cells were transfected with pVIM or pEGFP. VIM expression levels were determined by Western blotting at 24 h and 48 h after transfection. EGFP serves as the negative control and GAPDH as the loading control. E, Effect of VIM knockdown and overexpression on cell invasion as determined with a wound-healing assay. F, Quantification of the wound healing assay. G, Effect of VIM knockdown and overexpression on cell invasion, as determined with a Boyden chamber assay. H, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *p < 0.05; **p < 0.01. I, HOTAIR suppressed tumor growth and regulated VIM expression in nude mice. HeLa cells transfected with siHOTAIR or siNC and HeLa cells expressing either control shRNA or shHOTAIR were injected subcutaneously into the right flank of nude mice. After 20 days, mice were sacrificed and tumors were dissected and weighed. Representative photographs of xenografts were taken 20 days after injection of HeLa cells transfected with siHOTAIR or HeLa-KD cells. J, Quantification of tumor weight. Data are presented as means ± S.D. (n = 5). K, Western blotting of VIM protein expression in tumors excised from the mice indicated 20 days after injection.

Techniques Used: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Over Expression, Wound Healing Assay, Boyden Chamber Assay, shRNA, Injection

Inhibition of HOTAIR or overexpression of vimentin affects organization of the vimentin IF network. A, Representative confocal microscopy images showing the organization of vimentin IF in HeLa cells after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells B, Quantification of vimentin IF collapse in cells in A. C, Representative cells showing organization of vimentin IF under super-resolution microscopy after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells. D, Quantification of the diameter of vimentin IFs in the cells in C. (Scale bars: 30 μm in A, 5 μm in C). At least three independent experiments were performed under each condition with at least 100 cells (B) or five 15 × 15 μm images of different cells (D) quantified per experiment. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Figure Legend Snippet: Inhibition of HOTAIR or overexpression of vimentin affects organization of the vimentin IF network. A, Representative confocal microscopy images showing the organization of vimentin IF in HeLa cells after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells B, Quantification of vimentin IF collapse in cells in A. C, Representative cells showing organization of vimentin IF under super-resolution microscopy after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells. D, Quantification of the diameter of vimentin IFs in the cells in C. (Scale bars: 30 μm in A, 5 μm in C). At least three independent experiments were performed under each condition with at least 100 cells (B) or five 15 × 15 μm images of different cells (D) quantified per experiment. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Techniques Used: Inhibition, Over Expression, Confocal Microscopy, Knockdown, Super-Resolution Microscopy

Inhibition of HOTAIR leads to mitochondrial dysfunction. A, The concentration of UQCR in HeLa cells decreased after HOTAIR knockdown as measured using ELISA assays. B, Effect of HOTAIR knockdown on cellular ROS production as detected by flow cytometry analysis. C, Quantification of DCF fluorescence in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. D, Images of cellular glucose photographed under a confocal microscope. E, HOTAIR affects glucose uptake in HeLa cells. Cells had lower cellular glucose levels after HOTAIR knockdown. Glucose uptake was measured by FACS, following 0.5 h exposure to 2-NBDG (100 μm). F, Quantification of the fluorescence of cellular glucose in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. G, Electron microscopy images of (a) siNC cells and (b) siHOTAIR cells. Labels: M = mitochondria, n = nucleus. H, HOTAIR knockdown affects the mitochondrial membrane potential. Mitochondria were stained with MitoTracker Deep Red and representative images were obtained by confocal microscopy. I, HeLa cells were stained against JC-1 for flow cytometry after HOTAIR knockdown. There was a significant increase in the number of cells with green fluorescence (FL1 (R3)), indicating a decrease in the Δψ. J, The mean fluorescence intensity ratio (FL2/FL1) in HeLa cells after HOTAIR knockdown. CCCP was the positive control. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Figure Legend Snippet: Inhibition of HOTAIR leads to mitochondrial dysfunction. A, The concentration of UQCR in HeLa cells decreased after HOTAIR knockdown as measured using ELISA assays. B, Effect of HOTAIR knockdown on cellular ROS production as detected by flow cytometry analysis. C, Quantification of DCF fluorescence in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. D, Images of cellular glucose photographed under a confocal microscope. E, HOTAIR affects glucose uptake in HeLa cells. Cells had lower cellular glucose levels after HOTAIR knockdown. Glucose uptake was measured by FACS, following 0.5 h exposure to 2-NBDG (100 μm). F, Quantification of the fluorescence of cellular glucose in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. G, Electron microscopy images of (a) siNC cells and (b) siHOTAIR cells. Labels: M = mitochondria, n = nucleus. H, HOTAIR knockdown affects the mitochondrial membrane potential. Mitochondria were stained with MitoTracker Deep Red and representative images were obtained by confocal microscopy. I, HeLa cells were stained against JC-1 for flow cytometry after HOTAIR knockdown. There was a significant increase in the number of cells with green fluorescence (FL1 (R3)), indicating a decrease in the Δψ. J, The mean fluorescence intensity ratio (FL2/FL1) in HeLa cells after HOTAIR knockdown. CCCP was the positive control. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Techniques Used: Inhibition, Concentration Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Microscopy, Electron Microscopy, Membrane, Staining, Confocal Microscopy, Positive Control

Proposed model depicting the molecular mechanism of HOTAIR in regulating migration and invasion of HeLa cells. HOTAIR promotes cell migration and invasion in HeLa cells via different mechanisms. HOTAIR may serve as a molecular scaffold linking two distinct histone modification complexes to regulate hundreds of genes (12). HOTAIR regulates the expression and organization of vimentin. Our functional study demonstrated that vimentin contributes to the decreased migration and invasion capability of HeLa cells caused by inhibition of HOTAIR. The mitochondrial dysfunction caused by inhibition of HOTAIR may be another cause of the decreased migration and invasion capability of HeLa cells. The combination of all these mechanisms regulates the expression of hundreds of proteins and promotes cell migration and invasion. Vimentin may be a key molecule in HOTAIR-mediated oncogenic signaling.

Figure Legend Snippet: Proposed model depicting the molecular mechanism of HOTAIR in regulating migration and invasion of HeLa cells. HOTAIR promotes cell migration and invasion in HeLa cells via different mechanisms. HOTAIR may serve as a molecular scaffold linking two distinct histone modification complexes to regulate hundreds of genes (12). HOTAIR regulates the expression and organization of vimentin. Our functional study demonstrated that vimentin contributes to the decreased migration and invasion capability of HeLa cells caused by inhibition of HOTAIR. The mitochondrial dysfunction caused by inhibition of HOTAIR may be another cause of the decreased migration and invasion capability of HeLa cells. The combination of all these mechanisms regulates the expression of hundreds of proteins and promotes cell migration and invasion. Vimentin may be a key molecule in HOTAIR-mediated oncogenic signaling.

Techniques Used: Migration, Modification, Expressing, Functional Assay, Inhibition

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Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Functional effects of HOTAIR inhibition on HeLa cells. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h. HOTAIR knockdown efficiency was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. B, Apoptosis was determined by annexin V staining and flow cytometry. C, Apoptosis rate of HeLa cells at 48 h after transfection with siHOTAIR or siNC. D, Effect of HOTAIR knockdown on cell cycle progress. The percentage of cells in the G1 phase was significantly decreased whereas that in S phase and G2/M phase was increased after HOTAIR inhibition. E, HOTAIR knockdown in HeLa cells significantly inhibits cell growth. F, Effect of HOTAIR knockdown on cell invasion, as determined in a Boyden chamber assay. G, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. H, HOTAIR inhibition led to a significant reduction of cell migration as determined by a wound-healing assay. I, Quantification of the wound healing assay. Data are presented as means ± S.D. and represent results from three independent experiments. Statistically significant differences are indicated: *p < 0.05; **p < 0.01.

Article Snippet: Cell Culture and RNA Interference Human cervical cancer cell line HeLa, Chang Liver, human macrophage-like cell line U937, multiple myeloma cell line U266, lung cancer cell line H1299, hepatocellular carcinoma line HepG2 and gastric cancer cell line SGC7901 were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Functional Assay, Inhibition, Transfection, Knockdown, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Boyden Chamber Assay, Migration, Wound Healing Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Quantitative proteomic identification of HOTAIR-regulated proteins in HeLa cells. A, Workflow for the identification of HOTAIR-regulated proteins. HeLa cells were differentially labeled by growing them in medium containing light or heavy amino acids (SILAC). Proteins were extracted from the labeled cells 48 h after transfection with siHOTAIR or siNC, then equal amounts of protein from each sample were combined. The protein mix was separated by 12% SDS-PAGE and the resulting gel was cut into 30 sections. Each of the fractions was in-gel digested and analyzed via LC-MS/MS. B, Heatmap showing the expression of differentially expressed proteins after HOTAIR inhibition. C, PANTHER Protein Class ontology classification of the 170 proteins differentially expressed after HOTAIR expression silencing. HOTAIR-regulated proteins were classified into 22 classes.

Techniques: Labeling, Multiplex sample analysis, Transfection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: VIM contributes to the effects of HOTAIR knockdown. A, HeLa cells were transfected with siHOTAIR or siNC for 48 h and VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. B, HeLa cells were transfected with siVIM for 48 h, then VIM mRNA expression levels were determined by qRT-PCR. The expression level of VIM was normalized to GAPDH. C, Western blot analysis of VIM protein expression 48 h after transfection with siVIM (siVIM-I, siVIM-II and siVIM-III) or siNC. GAPDH was used as an internal control. D, HeLa cells were transfected with pVIM or pEGFP. VIM expression levels were determined by Western blotting at 24 h and 48 h after transfection. EGFP serves as the negative control and GAPDH as the loading control. E, Effect of VIM knockdown and overexpression on cell invasion as determined with a wound-healing assay. F, Quantification of the wound healing assay. G, Effect of VIM knockdown and overexpression on cell invasion, as determined with a Boyden chamber assay. H, Numbers of cells on the underside of the filter. Significantly enhanced invasion (p < 0.05) is indicated. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *p < 0.05; **p < 0.01. I, HOTAIR suppressed tumor growth and regulated VIM expression in nude mice. HeLa cells transfected with siHOTAIR or siNC and HeLa cells expressing either control shRNA or shHOTAIR were injected subcutaneously into the right flank of nude mice. After 20 days, mice were sacrificed and tumors were dissected and weighed. Representative photographs of xenografts were taken 20 days after injection of HeLa cells transfected with siHOTAIR or HeLa-KD cells. J, Quantification of tumor weight. Data are presented as means ± S.D. (n = 5). K, Western blotting of VIM protein expression in tumors excised from the mice indicated 20 days after injection.

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Over Expression, Wound Healing Assay, Boyden Chamber Assay, shRNA, Injection

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Inhibition of HOTAIR or overexpression of vimentin affects organization of the vimentin IF network. A, Representative confocal microscopy images showing the organization of vimentin IF in HeLa cells after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells B, Quantification of vimentin IF collapse in cells in A. C, Representative cells showing organization of vimentin IF under super-resolution microscopy after HOTAIR knockdown or VIM overexpression, or in HeLa-KD cells. D, Quantification of the diameter of vimentin IFs in the cells in C. (Scale bars: 30 μm in A, 5 μm in C). At least three independent experiments were performed under each condition with at least 100 cells (B) or five 15 × 15 μm images of different cells (D) quantified per experiment. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Techniques: Inhibition, Over Expression, Confocal Microscopy, Knockdown, Super-Resolution Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Inhibition of HOTAIR leads to mitochondrial dysfunction. A, The concentration of UQCR in HeLa cells decreased after HOTAIR knockdown as measured using ELISA assays. B, Effect of HOTAIR knockdown on cellular ROS production as detected by flow cytometry analysis. C, Quantification of DCF fluorescence in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. D, Images of cellular glucose photographed under a confocal microscope. E, HOTAIR affects glucose uptake in HeLa cells. Cells had lower cellular glucose levels after HOTAIR knockdown. Glucose uptake was measured by FACS, following 0.5 h exposure to 2-NBDG (100 μm). F, Quantification of the fluorescence of cellular glucose in HeLa cells. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01. G, Electron microscopy images of (a) siNC cells and (b) siHOTAIR cells. Labels: M = mitochondria, n = nucleus. H, HOTAIR knockdown affects the mitochondrial membrane potential. Mitochondria were stained with MitoTracker Deep Red and representative images were obtained by confocal microscopy. I, HeLa cells were stained against JC-1 for flow cytometry after HOTAIR knockdown. There was a significant increase in the number of cells with green fluorescence (FL1 (R3)), indicating a decrease in the Δψ. J, The mean fluorescence intensity ratio (FL2/FL1) in HeLa cells after HOTAIR knockdown. CCCP was the positive control. Data are presented as means ± S.D. and represent results from three independent experiments. *p < 0.05; **p < 0.01.

Techniques: Inhibition, Concentration Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Microscopy, Electron Microscopy, Membrane, Staining, Confocal Microscopy, Positive Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells *

doi: 10.1074/mcp.M114.043984

Figure Lengend Snippet: Proposed model depicting the molecular mechanism of HOTAIR in regulating migration and invasion of HeLa cells. HOTAIR promotes cell migration and invasion in HeLa cells via different mechanisms. HOTAIR may serve as a molecular scaffold linking two distinct histone modification complexes to regulate hundreds of genes (12). HOTAIR regulates the expression and organization of vimentin. Our functional study demonstrated that vimentin contributes to the decreased migration and invasion capability of HeLa cells caused by inhibition of HOTAIR. The mitochondrial dysfunction caused by inhibition of HOTAIR may be another cause of the decreased migration and invasion capability of HeLa cells. The combination of all these mechanisms regulates the expression of hundreds of proteins and promotes cell migration and invasion. Vimentin may be a key molecule in HOTAIR-mediated oncogenic signaling.

Techniques: Migration, Modification, Expressing, Functional Assay, Inhibition

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