Limits...
Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea.

Jadali A, Kwan KY - Biol Open (2016)

Bottom Line: In aging animals, components for active PI3K signaling are present but decrease in hair cells.Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death.These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA Stem Cell Research Center and Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.

No MeSH data available.


Related in: MedlinePlus

Proliferation and differentiation potential of iMOP cells after bFGF withdrawal. (A) Fluorescence intensity from DNA binding dye on iMOP cells cultured in the presence (n=3) or absence (n=3) of bFGF for 3 days. Fluorescence intensities are normalized to proliferating cultures to reflect changes in cell numbers. (B) Immunofluorescence images of EdU-labeled iMOP cells cultured in the presence or absence of bFGF for 3 days. (C) Percentages of EdU-labeled iMOP cells grown in the presence (n=5) or absence (n=5) of bFGF for 3 days. (D) Timeline for harvesting iMOP cells after growth factor withdrawal. (E) qPCR depicting relative transcript levels of MYO6, GFAP, and TUBB3 transcripts in proliferating (n=3) and differentiating iMOP cells cultured in absence bFGF for 3 days (n=3) and 7 days (n=3). Transcript levels are normalized to GAPDH and expressed as an average fold-change. (F-H) Representative immunofluorescence images from 7 day-differentiated iMOP cells for (F) MYO6 (n=3), (G) GFAP (n=3), and (H) TUBB3 (n=3). Data represented as mean±s.d.; *P<0.05, **P<1×10−2, ****P<1×10−4 by unpaired two-tailed Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920183&req=5

BIO016758F1: Proliferation and differentiation potential of iMOP cells after bFGF withdrawal. (A) Fluorescence intensity from DNA binding dye on iMOP cells cultured in the presence (n=3) or absence (n=3) of bFGF for 3 days. Fluorescence intensities are normalized to proliferating cultures to reflect changes in cell numbers. (B) Immunofluorescence images of EdU-labeled iMOP cells cultured in the presence or absence of bFGF for 3 days. (C) Percentages of EdU-labeled iMOP cells grown in the presence (n=5) or absence (n=5) of bFGF for 3 days. (D) Timeline for harvesting iMOP cells after growth factor withdrawal. (E) qPCR depicting relative transcript levels of MYO6, GFAP, and TUBB3 transcripts in proliferating (n=3) and differentiating iMOP cells cultured in absence bFGF for 3 days (n=3) and 7 days (n=3). Transcript levels are normalized to GAPDH and expressed as an average fold-change. (F-H) Representative immunofluorescence images from 7 day-differentiated iMOP cells for (F) MYO6 (n=3), (G) GFAP (n=3), and (H) TUBB3 (n=3). Data represented as mean±s.d.; *P<0.05, **P<1×10−2, ****P<1×10−4 by unpaired two-tailed Student's t-test.

Mentions: iMOP cultures allow for harvesting of a large number of otic fate restricted cells for RNA-seq. Proliferating iMOP cells were grown in suspension as colony-forming otic cells, known as otospheres. To initiate differentiation into hair cells and supporting cells bFGF, the sole growth factor in the media, was withdrawn from iMOP cultures (Jadali et al., 2015). Two methods were employed to monitor cell cycle arrest. First, a fluorescence-based assay was used as a measure of cell numbers to determine the proliferative status of the cultures. iMOP cells were cultured either in the presence or absence of bFGF for 3 days before labeling with CyQuant direct nucleic acid stain, a cell permeable fluorescent DNA dye to assay for total DNA content. Emitted fluorescence from the DNA bound dye served as an index of total cell numbers. Cultures grown in the absence of bFGF showed a significant decrease in cell numbers compared to proliferating cultures (P<1×10−2) (Fig. 1A).Fig. 1.


Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea.

Jadali A, Kwan KY - Biol Open (2016)

Proliferation and differentiation potential of iMOP cells after bFGF withdrawal. (A) Fluorescence intensity from DNA binding dye on iMOP cells cultured in the presence (n=3) or absence (n=3) of bFGF for 3 days. Fluorescence intensities are normalized to proliferating cultures to reflect changes in cell numbers. (B) Immunofluorescence images of EdU-labeled iMOP cells cultured in the presence or absence of bFGF for 3 days. (C) Percentages of EdU-labeled iMOP cells grown in the presence (n=5) or absence (n=5) of bFGF for 3 days. (D) Timeline for harvesting iMOP cells after growth factor withdrawal. (E) qPCR depicting relative transcript levels of MYO6, GFAP, and TUBB3 transcripts in proliferating (n=3) and differentiating iMOP cells cultured in absence bFGF for 3 days (n=3) and 7 days (n=3). Transcript levels are normalized to GAPDH and expressed as an average fold-change. (F-H) Representative immunofluorescence images from 7 day-differentiated iMOP cells for (F) MYO6 (n=3), (G) GFAP (n=3), and (H) TUBB3 (n=3). Data represented as mean±s.d.; *P<0.05, **P<1×10−2, ****P<1×10−4 by unpaired two-tailed Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920183&req=5

BIO016758F1: Proliferation and differentiation potential of iMOP cells after bFGF withdrawal. (A) Fluorescence intensity from DNA binding dye on iMOP cells cultured in the presence (n=3) or absence (n=3) of bFGF for 3 days. Fluorescence intensities are normalized to proliferating cultures to reflect changes in cell numbers. (B) Immunofluorescence images of EdU-labeled iMOP cells cultured in the presence or absence of bFGF for 3 days. (C) Percentages of EdU-labeled iMOP cells grown in the presence (n=5) or absence (n=5) of bFGF for 3 days. (D) Timeline for harvesting iMOP cells after growth factor withdrawal. (E) qPCR depicting relative transcript levels of MYO6, GFAP, and TUBB3 transcripts in proliferating (n=3) and differentiating iMOP cells cultured in absence bFGF for 3 days (n=3) and 7 days (n=3). Transcript levels are normalized to GAPDH and expressed as an average fold-change. (F-H) Representative immunofluorescence images from 7 day-differentiated iMOP cells for (F) MYO6 (n=3), (G) GFAP (n=3), and (H) TUBB3 (n=3). Data represented as mean±s.d.; *P<0.05, **P<1×10−2, ****P<1×10−4 by unpaired two-tailed Student's t-test.
Mentions: iMOP cultures allow for harvesting of a large number of otic fate restricted cells for RNA-seq. Proliferating iMOP cells were grown in suspension as colony-forming otic cells, known as otospheres. To initiate differentiation into hair cells and supporting cells bFGF, the sole growth factor in the media, was withdrawn from iMOP cultures (Jadali et al., 2015). Two methods were employed to monitor cell cycle arrest. First, a fluorescence-based assay was used as a measure of cell numbers to determine the proliferative status of the cultures. iMOP cells were cultured either in the presence or absence of bFGF for 3 days before labeling with CyQuant direct nucleic acid stain, a cell permeable fluorescent DNA dye to assay for total DNA content. Emitted fluorescence from the DNA bound dye served as an index of total cell numbers. Cultures grown in the absence of bFGF showed a significant decrease in cell numbers compared to proliferating cultures (P<1×10−2) (Fig. 1A).Fig. 1.

Bottom Line: In aging animals, components for active PI3K signaling are present but decrease in hair cells.Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death.These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA Stem Cell Research Center and Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.

No MeSH data available.


Related in: MedlinePlus