bioimagesUnknownBharat JoshiThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201207089JCB

Cav1Wt-expressing MDA-435 stable cells were labelled for F-actin. Cellswere fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to incubation with Alexa568–phalloidin. Coverslips were mounted in Airvol (Air Products, Inc., Allentown, PA) and images collected with the 100x planapochromat objective (NA 1.35) of an FV1000 Olympus confocal microscope.

Cav1Y14D-expressing MDA-435 stable cells were labelled for F-actin. Cellswere fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to incubation with Alexa568–phalloidin. Coverslips were mounted in Airvol (Air Products, Inc., Allentown, PA) and images collected with the 100x planapochromat objective (NA 1.35) of an FV1000 Olympus confocal microscope.

Transiently transfected Cav1-/- immortalized mouse embryonic fibroblasts (MEFs) expressing Cav1Y14D construct were fixed in the presence of ruthenium red to highlight the cell surface, processed by conventional methods for Epon embedding. Cav1-/- cells were sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x.

Cav1Y14F-expressing MDA-435 stable cells were labelled for F-actin. Cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to incubation with Alexa568–phalloidin. Coverslips were mounted in Airvol (Air Products, Inc., Allentown, PA) and images collected with the 100x planapochromat objective (NA 1.35) of an FV1000 Olympus confocal microscope.

Transiently transfected Cav1-/- immortalized mouse embryonic fibroblasts (MEFs) expressing Cav1wt construct were fixed in the presence of ruthenium red to highlight the cell surface, processed by conventional methods for Epon embedding. Cav1-/- cells were sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x.

Transiently transfected Cav1-/- immortalized mouse embryonic fibroblasts (MEFs) expressing Cav1Y14R construct were fixed in the presence of ruthenium red to highlight the cell surface, processed by conventional methods for Epon embedding. Cav1-/- cells were sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x.

DsRed-expressing MDA-435 stable cells were labelled for F-actin. Cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to incubation with Alexa568–phalloidin. Coverslips were mounted in Airvol (Air Products, Inc., Allentown, PA) and images collected with the 100x planapochromat objective (NA 1.35) of an FV1000 Olympus confocal microscope.

MDA-435 stable cells expressing Cav1Wt (wild-type) were fixed in the presence of ruthenium red to highlight the cell surface and processed by conventional methods for Epon embedding, sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x. For quantification of caveolae density in MDA-435 cell lines, sections were cut perpendicular to the cell surface (vertical sections) to ensure random sampling of the entire plasma membrane.

Cav1-/- immortalized mouse embryonic fibroblasts (MEFs) fixed in the presence of ruthenium red to highlight the cell surface, processed by conventional methods for Epon embedding. Cav1-/- cells were sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x.

MDA-435 stable cells expressing Cav1Y14R were fixed in the presence of ruthenium red to highlight the cell surface processed by conventional methods for Epon embedding, sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x. For quantification of caveolae density in MDA-435 cell lines, sections were cut perpendicular to the cell surface (vertical sections) to ensure random sampling of the entire plasma membrane.

Cav1Y14R-expressing MDA-435 stable cells were labelled for F-actin. Cellswere fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to incubation with Alexa568–phalloidin. Coverslips were mounted in Airvol (Air Products, Inc., Allentown, PA) and images collected with the 100x planapochromat objective (NA 1.35) of an FV1000 Olympus confocal microscope.

Transiently transfected Cav1-/- immortalized mouse embryonic fibroblasts (MEFs) expressing Cav1Y14F construct were fixed in the presence of ruthenium red to highlight the cell surface, processed by conventional methods for Epon embedding. Cav1-/- cells were sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x.

MDA-435 stable cells expressing Cav1Y14D were fixed in the presence of ruthenium red to highlight the cell surface processed by conventional methods for Epon embedding, sectioned parallel to the substratum and images captured using a Jeol 1011 TEM equipped with a Morada cooled CCD camera and Olympus Soft Imaging Solutions Gmbh, Germany with AnalySIS software at a magnification of 15,000–20,000x. For quantification of caveolae density in MDA-435 cell lines, sections were cut perpendicular to the cell surface (vertical sections) to ensure random sampling of the entire plasma membrane.

bioimagesImage 137621 (Figure 2 - B)Supplemental Figure 2Image 138074 (Figure 1 - B)Image 138072 (Figure 1 - B)Image 137625 (Figure 2 - B)Image 138093 (Supplemental Figure 2 - A)Figure 1 - BImage 137623 (Figure 2 - B)Image 138091 (Supplemental Figure 2 - A)Image 138090 (Supplemental Figure 2 - A)Image 138076 (Figure 1 - B)Supplemental Figure 2 - AImage 138071 (Figure 1 - B)Image 138073 (Figure 1 - B)Image 137624 (Figure 2 - B)Image 138075 (Figure 1 - B)Figure 1Image 138094 (Supplemental Figure 2 - A)Image 137622 (Figure 2 - B)Image 138092 (Supplemental Figure 2 - A)Figure 2Figure 2 - BBharat JoshiIvan R. NabiMichele BastianiRobert G. PartonScott S. StrugnellCecile BoscherfalsePhosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulationCaveolin-1 (Cav1) is an essential component of caveolae whose Src kinase-dependent phosphorylation on tyrosine 14 (Y14) is associated with regulation of focal adhesion dynamics. However, the relationship between these disparate functions remains to be elucidated. Caveola biogenesis requires expression of both Cav1 and cavin-1, but Cav1Y14 phosphorylation is dispensable. In this paper, we show that Cav1 tyrosine phosphorylation induces caveola biogenesis via actin-dependent mechanotransduction and inactivation of the Egr1 (early growth response-1) transcription factor, relieving inhibition of endogenous<jats:italic>Cav1</jats:italic>and<jats:italic>cavin-1</jats:italic>genes. Cav1 phosphorylation reduces Egr1 binding to Cav1 and cavin-1 promoters and stimulates their activity. In MDA-231 br2012-10-22T11:23:45Z2018-11-29T11:23:45Z2018-11-29T11:23:45ZS-JCBD-20120708910.1083/jcb.201207089