Comparison of thickness and asymmetry measurements across tomogram reconstruction methods for control vs cholesterol-depleted cells. (A) Fluorometric assay results for validation of cholesterol depletion (see Materials and methods). The table shows total cell counts, soluble protein concentration, and cholesterol levels in each condition, along with normalized values (cholesterol per protein [µg/µg] and µg cholesterol per cell). When normalized to protein content or per cell, treatment with 10 mM MBCD reduced cholesterol levels by 40%. (B and C) Central slices from tomograms reconstructed with WBP algorithm from cryoCARE denoised (left) or unprocessed frames (right), showing control (B) and cholesterol-depleted (C) HEK293 cells in contact. Two PMs (PM1, PM2) are visible at the cell–cell interfaces. The cryoCARE-denoised tomogram shown in B is the same as in Fig. 6 D, shown for compa. Scale bars, 100 nm. (D and E) Mean intensity profiles for PM1 extracted from WBP tomograms from unprocessed frames in control (D) and cholesterol-depleted (E) cells. Vertical lines mark the mean positions of paired measurement points used for thickness calculation. For details on the asymmetry score calculation, see Materials and methods. (F and G) Normalized thickness distributions from WBP tomograms from unprocessed frames for control (F) and cholesterol-depleted (G) cells. (H and I) Thickness-binned asymmetry distributions for control (H) and cholesterol-depleted (I) PMs derived from WBP tomograms, expressed as percentage asymmetry where 1.0 = 0% asymmetry (or perfect symmetry) and values >1.0 indicate increasing asymmetry [(score -1.0) × 100%].