LCBs are integral components of the nuclear membrane in human cells. (a) Chemical structure of the fluorescent probes used in our studies. DHS, dihydrosphingosine; Cer, ceramide; NBD, nitrobenzoxadiazole. (b) Live-cell microscopy of HeLa cells expressing mCherry-Farnesyl-5 (red, plasma membrane) and incubated with 1 µM DHS-NBD (green) for 20 min. Scale bar in the left image, 10 µm, and in zoomed images, scale bar, 2 µm. NE indicates NE. Fluorescence intensity across the white dotted lines is shown below. (c) Live-cell microscopy of HeLa cells expressing mCherry-Farnesyl-5 and incubated with 1 µM C18-Cer-NBD (green) for 20 min. Scale bar in the left image, 10 µm; in zoomed images, scale bar, 2 µm. PM, plasma membrane. Fluorescence intensity across the white dotted lines is shown below. (d–g) Live-cell microscopy of HeLa cells incubated with 1 µM DHS-NBD (green) for 20 min. In d, cells express Sec61-mCherry (NE and ER); in e, cells express mCherry-Golgi-7 (Golgi apparatus); in f, cells express LAMP1-mCherry (lysosome); and in g, cell express mCherry-mito-7 (mitochondria). Scale bar in the left image, 10 µm; in zoomed images, scale bar, 2 µm. Fluorescence intensity across the white dotted lines is shown below. (h) DIC images of a T cell and purified nucleus. Scale bar, 2 µm. (i) Western blot analysis of the different organelle markers in whole-cell extracts and isolated nuclei from T cells. (j and k) HPLC-MS/MS analysis of ceramides (j) and LCBs (k) of primary T cells and isolated nuclei from the T cells. Error bars represent standard deviations (n = 3 independent samples from human blood, >10⁶ cells or nuclei analyzed per sample). *P < 1e-4, unpaired t test. NS, P value >0.05. HPLC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry. Source data are available for this figure: SourceData F5.