Fig. 5: Cav1 Y14 is needed for LD expansion-induced caveolae reorganization.
From: Plasma membrane remodeling determines adipocyte expansion and mechanical adaptability
a Targeting strategy for producing the Y14F conditional allele. The loxP sites are marked with triangles. TK: thymidine kinase gene for negative selection of ES clones by resistance to G418/ganciclovir. PGK-neo: neomycine resistance gene under the control of the phosphoglycerate kinase promoter. b Western blot analysis of Cav1 levels and phosphorylation in visceral WAT. Adipose tissue was treated with H2O2+Na3VO4 to induce Y14 phosphorylation and contralateral depots used as controls, demonstrating lack of phosphorylation in Y14F animals. An unspecific band of 25 kDa was present in all WAT samples (arrow), and thus samples were additionally subjected to immunoprecipitation with an antiCav1 antibody. Arrowhead marks specific Cav1 band height. c Representative WB of total and pCav1 in +/+ and Y14F/Y14F in whole lysates from in vitro differentiated 3T3-L1 adipocytes, genome-edited to exclusively express either wild type Cav1 or Y14F Cav1 mutant. d–g caveolae counts per µm of cell perimeter for total caveolae (d), single-pit caveolae (e), caveolae in rosettes (f) and cavicles (g). d–g Wilcoxon signed rank test p values from left to right: 0.004, 0.123, 0.002, 0.138. Sample size=64+/+ and 69 Y14F/Y14F cells. h–j Representative images of adipocyte caveolae at long (h), medium (I), and short (j) PM-LD distances. Scale bar: 0.5 µm. k–n Caveolae density measured as caveolae/µm of cell perimeter for total caveolae (k), rosette caveolae (l), cavicle (m), and single-pits (n). Sample size=69 cells. p values shown for the null hypothesis slope=0. o–r Spatial concentration of caveolae measured as caveolae/µm2 for total caveolae (o), rosette caveolae (p), cavicles (q), and single-pits (r) as a function of median PM-LD distance. Each dot is an individual cell. Sample size=69 cells. p values shown for the null hypothesis slope=0. For k–r error bands show 95% confidence interval for the estimation of the mean (line). p-value codes: * <0.05, **<0.01, ***<0.001. All boxplots show first (Q1), second (median) and third (Q3) quantiles, and whiskers extend from Q1-1.5·IQR to Q3+1.5×IQR. All tests are two-sided. Source data provided as a SourceData.zip.
