Figure 7.
LCBs are synthesized during S and G2 phases in human cells. (a) HPLC-MS/MS analysis of LCBs and ceramides of RPE-1 cell culture in 10% FBS. Error bars represent the standard deviation (n = 3 biological replicates, >106 cells analyzed in each experiment). (b) Representative images of RPE-1 cells in 10% FBS (Asynchronous, Asyn) or arrested in G1 with 2 µM palbociclib, in the S phase with 2 µM thymidine, or in late G2 with 10 µM RO-3306. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. FACS profiles are shown below. (c) Area of nuclei of RPE-1 cells (n = 200) as in b. The averaged calculated radius is shown below. *P < 1e-4, one-way ANOVA. (d) HPLC-MS/MS analysis of LCBs and ceramides in RPE-1 arrested as in b. Columns represent experiments (n = 3 biological replicates, >106 cells analyzed in each experiment), and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; Sph, sphingosine. (e) Average log2 ratios of the lipid levels in G1- or G2-arrested cells are shown. Error bars represent standard deviations (n = 3 biological replicates). (f) Representative images of RPE-1 cells in LD-FBS (Asynchronous, Asyn) or arrested in G1 with 2 µM palbociclib, in the S phase with 2 µM thymidine, or in late G2 with 10 µM RO-3306. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. FACS profiles are shown below. (g) Area of nuclei of RPE-1 cells (n = 200 nuclei) as in f. The averaged calculated radius is shown below. Nuclei with abnormal morphology are not included in G2 arrest. *P < 1e-4, one-way ANOVA. (h) Percentage of cells with abnormal nuclear morphology. Error bars represent the standard deviation of 3 biological replicates (each replicate, n >100 cells). *P < 1e-4, unpaired t test. (i) HPLC-MS/MS analysis of LCBs and ceramides in RPE-1 arrested as in f. Log2 ratios of the lipid levels relative to asynchronous cells are shown. Columns represent experiments, and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; Sph, sphingosine. Scale bar, 5 µm. (j) Quantification of total LCBs and ceramides in G1- or G2-arrested RPE-1 cells in LD-FBS. Error bars represent the standard deviations (n = 3 biological replicates). (k) Representative images of RPE-1 cells in lipid-depleted media arrested in G2 in the presence of 1 µM Sph. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. (l) Percentage of cells with abnormal nuclear morphology. Error bars represent the standard deviation of three independent biological replicates (each replicate, n > 100 cells). Cells were arrested in G2 alone or with 1 µM DHS, 1 µM Sph, or 1 µM ceramide. *P < 1e-4, one-way ANOVA. HPLC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry.

LCBs are synthesized during S and G2 phases in human cells. (a) HPLC-MS/MS analysis of LCBs and ceramides of RPE-1 cell culture in 10% FBS. Error bars represent the standard deviation (n = 3 biological replicates, >106 cells analyzed in each experiment). (b) Representative images of RPE-1 cells in 10% FBS (Asynchronous, Asyn) or arrested in G1 with 2 µM palbociclib, in the S phase with 2 µM thymidine, or in late G2 with 10 µM RO-3306. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. FACS profiles are shown below. (c) Area of nuclei of RPE-1 cells (n = 200) as in b. The averaged calculated radius is shown below. *P < 1e-4, one-way ANOVA. (d) HPLC-MS/MS analysis of LCBs and ceramides in RPE-1 arrested as in b. Columns represent experiments (n = 3 biological replicates, >106 cells analyzed in each experiment), and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; Sph, sphingosine. (e) Average log2 ratios of the lipid levels in G1- or G2-arrested cells are shown. Error bars represent standard deviations (n = 3 biological replicates). (f) Representative images of RPE-1 cells in LD-FBS (Asynchronous, Asyn) or arrested in G1 with 2 µM palbociclib, in the S phase with 2 µM thymidine, or in late G2 with 10 µM RO-3306. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. FACS profiles are shown below. (g) Area of nuclei of RPE-1 cells (n = 200 nuclei) as in f. The averaged calculated radius is shown below. Nuclei with abnormal morphology are not included in G2 arrest. *P < 1e-4, one-way ANOVA. (h) Percentage of cells with abnormal nuclear morphology. Error bars represent the standard deviation of 3 biological replicates (each replicate, n >100 cells). *P < 1e-4, unpaired t test. (i) HPLC-MS/MS analysis of LCBs and ceramides in RPE-1 arrested as in f. Log2 ratios of the lipid levels relative to asynchronous cells are shown. Columns represent experiments, and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; Sph, sphingosine. Scale bar, 5 µm. (j) Quantification of total LCBs and ceramides in G1- or G2-arrested RPE-1 cells in LD-FBS. Error bars represent the standard deviations (n = 3 biological replicates). (k) Representative images of RPE-1 cells in lipid-depleted media arrested in G2 in the presence of 1 µM Sph. Blue, Hoechst 33342 (DNA); and green, anti-lamin B1. Scale bar, 5 µm. (l) Percentage of cells with abnormal nuclear morphology. Error bars represent the standard deviation of three independent biological replicates (each replicate, n > 100 cells). Cells were arrested in G2 alone or with 1 µM DHS, 1 µM Sph, or 1 µM ceramide. *P < 1e-4, one-way ANOVA. HPLC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry.

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