HeLa (Cilengitide treatment or MBCD treatment) PREM membrane images supporting "The structure and spontaneous curvature of clathrin lattices at the plasma membrane"

This dataset is part of a collection of platinum replica electron microscopy data ( https://doi.org/10.25444/nhlbi.c.5334212) from the manuscript entitled:<br>"The structure and spontaneous curvature of clathrin lattices at the plasma membrane" by Sochacki et al. 2021 (https://doi.org/10.1016/j.devcel.2021.03.017).<br><br>Figure 4 of this manuscript performs several drug treatments on HeLa cells to understand their impact on clathrin structure. Treatments include DMSO, actin inhibiting drugs (https://doi.org/10.25444/nhlbi.14204363; Latrunculin A, Jasplikinolide, Cytochalasin D), cholesterol removal (MBCD), and selective integrin blocking (cilengitide).<br><br>All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm). <br><br>HeLa cells were treated with drugs to remove cholesterol (methyl-β-cyclodextrin) or block the binding of aVB5 integrin (Cilengitide). Removing cholesterol caused an increase in flat clathrin while blocking integrins caused a decrease in flat clathrin. Additionally, MBCD treated cells were incubated 5 minutes after unroofing in minimal media to find if the flat clathrin could spontaneously curve. All of these data can be compared to untreated HeLa cells uploaded into a different dataset.HeLa (ATCC® CCL-2™, female human cervix epithelial, adenocarcinoma) cells were grown in DMEM media supplemented with 10% fetal bovine serum , Glutamax, and sodium pyruvate. They were grown on Poly-lysine coated coverslips for one day prior to unroofing. They were treated with Cilengitide (10 µM, Sigma-Aldrich, SML1594) or methyl-β-cyclodextrin (9.5 µM, Sigma-Aldrich C4555) added to growth media at normal growth conditions for one hour prior to unroofing/fixation.They were rinsed briefly with unroofing buffer (30mM HEPES, 70mM KCl, 5mM MgCl2, 3mM EGTA at pH 7.4) prior to unroofing. They were unroofed by brief sonication pulse in the presence of 0.5% paraformaldehyde or direct syringe flow in the presence of 2% paraformaldehyde. The coverslips were then moved immediately into unroofing buffer containing 2% glutaraldehyde. For the methyl-β-cyclodextrin (MBCD) 5 minute data, the membranes were unroofed with no fixative present. They were then incubated in stabilization buffer containing MBCD for 5 minutes before being fixed with glutaraldehyde. <br>Cells were kept in glutaraldehyde at 4 degs C until ready for staining and dehydration; less than two days. The cells were stained with 0.1% tannic acid for 20 minutes, rinsed, then stained with 0.1% uranyl acetate for 20 minutes and dehydrated slowly through a series of increasing ethanol concentration to 100% ethanol. The coverslips were then dried with critical point drying and rotary shadowed with platinum and carbon. The replicas were released from the coverslip with hydrofluoric acid and placed onto a TEM grid for imaging. TEM imaging of replicas was performed on a JEOL 1400 equipped with SerialEM freeware for montaging.