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Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

DeOcesano-Pereira C, Amaral MS, Parreira KS, Ayupe AC, Jacysyn JF, Amarante-Mendes GP, Reis EM, Verjovski-Almeida S - Nucleic Acids Res. (2014)

Bottom Line: These effects were abrogated in the presence of INXS knockdown.Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis.BCL-XS protein accumulation was detected upon INXS overexpression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil.

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Overexpression of INXS induces BCL-XS mRNA and protein and promotes apoptosis. (A) The levels of BCL-XS (black) and BCL-XL (red) mRNA isoforms were measured by RT-qPCR in 786-O kidney tumor cells 24 h after transient transfection with increasing amounts of pCEP4-INXS plasmid (INXS lncRNA levels = blue bars). All expression levels are shown as relative abundance with respect to the endogenous INXS in wild-type cells. (See Supplementary Figure S6A and F for similar effects on the MCF7 and PC3 cell lines.) (B) The increase in BCL-XS/BCL-XL ratio is dependent on the extent of INXS overexpression. (C) Total BCL-X mRNA does not change upon INXS overexpression. (D) Augmented apoptosis upon transfection of 786-O cells with increasing amounts of pCEP-INXS plasmid, as detected by flow cytometry using double labeling with Annexin V FITC (AV FITC, x-axis) and propidium iodide (PI, y-axis). The percentage of cells that were labeled with AV FITC is shown in the quadrants marked with blue broken lines. (See Supplementary Figure S6D and I for similar effects on the MCF7 and PC3 cell lines.) (E) The results from (D) are shown as the fraction of labeled cells relative to the total. (F) Western blot detects the BCL-XS protein isoform upon INXS overexpression. Antibody anti-BCL-X was used for immunoprecipitation of 786-O cell lysates, and the IP fraction was analyzed by western blot with the same antibody. Three independent replicate transfection experiments are shown. (G) Densitometric intensity ratio between BCL-XS and BCL-XL signals from the data on panel F (the background intensity signal was used as a proxy for BCL-XS in the controls). (H) Caspases 3, 7 and 9 are activated upon INXS overexpression in 786-O cell line, while caspase 8 is not affected. (See Supplementary Figure S9 for detection of active caspase 3 by immunofluorescence microscopy.) In all panels except in D and F, the data are the mean ± SD of three independent experiments. **(P <0.01) and ***(P <0.001).
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Figure 6: Overexpression of INXS induces BCL-XS mRNA and protein and promotes apoptosis. (A) The levels of BCL-XS (black) and BCL-XL (red) mRNA isoforms were measured by RT-qPCR in 786-O kidney tumor cells 24 h after transient transfection with increasing amounts of pCEP4-INXS plasmid (INXS lncRNA levels = blue bars). All expression levels are shown as relative abundance with respect to the endogenous INXS in wild-type cells. (See Supplementary Figure S6A and F for similar effects on the MCF7 and PC3 cell lines.) (B) The increase in BCL-XS/BCL-XL ratio is dependent on the extent of INXS overexpression. (C) Total BCL-X mRNA does not change upon INXS overexpression. (D) Augmented apoptosis upon transfection of 786-O cells with increasing amounts of pCEP-INXS plasmid, as detected by flow cytometry using double labeling with Annexin V FITC (AV FITC, x-axis) and propidium iodide (PI, y-axis). The percentage of cells that were labeled with AV FITC is shown in the quadrants marked with blue broken lines. (See Supplementary Figure S6D and I for similar effects on the MCF7 and PC3 cell lines.) (E) The results from (D) are shown as the fraction of labeled cells relative to the total. (F) Western blot detects the BCL-XS protein isoform upon INXS overexpression. Antibody anti-BCL-X was used for immunoprecipitation of 786-O cell lysates, and the IP fraction was analyzed by western blot with the same antibody. Three independent replicate transfection experiments are shown. (G) Densitometric intensity ratio between BCL-XS and BCL-XL signals from the data on panel F (the background intensity signal was used as a proxy for BCL-XS in the controls). (H) Caspases 3, 7 and 9 are activated upon INXS overexpression in 786-O cell line, while caspase 8 is not affected. (See Supplementary Figure S9 for detection of active caspase 3 by immunofluorescence microscopy.) In all panels except in D and F, the data are the mean ± SD of three independent experiments. **(P <0.01) and ***(P <0.001).

Mentions: To test the effect of INXS ectopic overexpression on BCL-X alternative splicing, 786-O cells were transiently transfected with increasing concentrations of a pCEP4-INXS vector; after 24 h, the transfected 786-O cells showed a 15- to 40-fold increase in INXS compared with the endogenous INXS lncRNA expression level in untransfected cells (Figure 6A, blue bars). The mRNA abundance of the BCL-XL isoform was reduced up to 4-fold in 786-O cells transfected with 3 μg of plasmid compared with wild-type cells (Figure 6A, red bars). Interestingly, BCL-XS mRNA expression showed a 20-fold increase relative to that of wild-type (Figure 6A, black bars). It is noteworthy that the relative abundance ratio of BCL-XS/BCL-XL was markedly increased, up to ∼60-fold (Figure 6B), from 0.06 in untransfected cells to 3.3 in cells with the highest level of INXS lncRNA overexpression; no change in the total BCL-X mRNA (Figure 6C) was observed. At the highest INXS lncRNA levels, the pro-apoptotic BCL-XS isoform was clearly predominant (∼80% of all BCL-X mRNA in the cell).


Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

DeOcesano-Pereira C, Amaral MS, Parreira KS, Ayupe AC, Jacysyn JF, Amarante-Mendes GP, Reis EM, Verjovski-Almeida S - Nucleic Acids Res. (2014)

Overexpression of INXS induces BCL-XS mRNA and protein and promotes apoptosis. (A) The levels of BCL-XS (black) and BCL-XL (red) mRNA isoforms were measured by RT-qPCR in 786-O kidney tumor cells 24 h after transient transfection with increasing amounts of pCEP4-INXS plasmid (INXS lncRNA levels = blue bars). All expression levels are shown as relative abundance with respect to the endogenous INXS in wild-type cells. (See Supplementary Figure S6A and F for similar effects on the MCF7 and PC3 cell lines.) (B) The increase in BCL-XS/BCL-XL ratio is dependent on the extent of INXS overexpression. (C) Total BCL-X mRNA does not change upon INXS overexpression. (D) Augmented apoptosis upon transfection of 786-O cells with increasing amounts of pCEP-INXS plasmid, as detected by flow cytometry using double labeling with Annexin V FITC (AV FITC, x-axis) and propidium iodide (PI, y-axis). The percentage of cells that were labeled with AV FITC is shown in the quadrants marked with blue broken lines. (See Supplementary Figure S6D and I for similar effects on the MCF7 and PC3 cell lines.) (E) The results from (D) are shown as the fraction of labeled cells relative to the total. (F) Western blot detects the BCL-XS protein isoform upon INXS overexpression. Antibody anti-BCL-X was used for immunoprecipitation of 786-O cell lysates, and the IP fraction was analyzed by western blot with the same antibody. Three independent replicate transfection experiments are shown. (G) Densitometric intensity ratio between BCL-XS and BCL-XL signals from the data on panel F (the background intensity signal was used as a proxy for BCL-XS in the controls). (H) Caspases 3, 7 and 9 are activated upon INXS overexpression in 786-O cell line, while caspase 8 is not affected. (See Supplementary Figure S9 for detection of active caspase 3 by immunofluorescence microscopy.) In all panels except in D and F, the data are the mean ± SD of three independent experiments. **(P <0.01) and ***(P <0.001).
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Figure 6: Overexpression of INXS induces BCL-XS mRNA and protein and promotes apoptosis. (A) The levels of BCL-XS (black) and BCL-XL (red) mRNA isoforms were measured by RT-qPCR in 786-O kidney tumor cells 24 h after transient transfection with increasing amounts of pCEP4-INXS plasmid (INXS lncRNA levels = blue bars). All expression levels are shown as relative abundance with respect to the endogenous INXS in wild-type cells. (See Supplementary Figure S6A and F for similar effects on the MCF7 and PC3 cell lines.) (B) The increase in BCL-XS/BCL-XL ratio is dependent on the extent of INXS overexpression. (C) Total BCL-X mRNA does not change upon INXS overexpression. (D) Augmented apoptosis upon transfection of 786-O cells with increasing amounts of pCEP-INXS plasmid, as detected by flow cytometry using double labeling with Annexin V FITC (AV FITC, x-axis) and propidium iodide (PI, y-axis). The percentage of cells that were labeled with AV FITC is shown in the quadrants marked with blue broken lines. (See Supplementary Figure S6D and I for similar effects on the MCF7 and PC3 cell lines.) (E) The results from (D) are shown as the fraction of labeled cells relative to the total. (F) Western blot detects the BCL-XS protein isoform upon INXS overexpression. Antibody anti-BCL-X was used for immunoprecipitation of 786-O cell lysates, and the IP fraction was analyzed by western blot with the same antibody. Three independent replicate transfection experiments are shown. (G) Densitometric intensity ratio between BCL-XS and BCL-XL signals from the data on panel F (the background intensity signal was used as a proxy for BCL-XS in the controls). (H) Caspases 3, 7 and 9 are activated upon INXS overexpression in 786-O cell line, while caspase 8 is not affected. (See Supplementary Figure S9 for detection of active caspase 3 by immunofluorescence microscopy.) In all panels except in D and F, the data are the mean ± SD of three independent experiments. **(P <0.01) and ***(P <0.001).
Mentions: To test the effect of INXS ectopic overexpression on BCL-X alternative splicing, 786-O cells were transiently transfected with increasing concentrations of a pCEP4-INXS vector; after 24 h, the transfected 786-O cells showed a 15- to 40-fold increase in INXS compared with the endogenous INXS lncRNA expression level in untransfected cells (Figure 6A, blue bars). The mRNA abundance of the BCL-XL isoform was reduced up to 4-fold in 786-O cells transfected with 3 μg of plasmid compared with wild-type cells (Figure 6A, red bars). Interestingly, BCL-XS mRNA expression showed a 20-fold increase relative to that of wild-type (Figure 6A, black bars). It is noteworthy that the relative abundance ratio of BCL-XS/BCL-XL was markedly increased, up to ∼60-fold (Figure 6B), from 0.06 in untransfected cells to 3.3 in cells with the highest level of INXS lncRNA overexpression; no change in the total BCL-X mRNA (Figure 6C) was observed. At the highest INXS lncRNA levels, the pro-apoptotic BCL-XS isoform was clearly predominant (∼80% of all BCL-X mRNA in the cell).

Bottom Line: These effects were abrogated in the presence of INXS knockdown.Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis.BCL-XS protein accumulation was detected upon INXS overexpression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil.

Show MeSH
Related in: MedlinePlus