Figure 4.
LCB synthesis takes place upon entry into the cell cycle. (a) DNA content of yeast cells (n = 30,000) after release from a pheromone-induced G1 arrest without or with myriocin (2.5 µM) at indicated time points. The asterisk denotes the beginning of the S phase. (b) Western blot analysis of mitotic Clb2 levels after release from G1 arrest. The PSTAIR antibody was used as a loading control. Relative levels of Clb2 quantified with ImageJ are shown under the western mages. The percentage of budded cells quantified by visual inspection of DIC images are shown below (n = 200). (c) HPLC-MS/MS analysis of LCB and ceramides in yeast cells harvested at indicated time points after release from a G1 arrest. Log2 ratios of the lipid levels relative to time 0 min are shown. Columns represent experiments, and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; PHS, phytosphingosine. (d) Plots of the total lipid classes as a function of time of the lipidome data presented in Fig. 2 c. *P < 1 e-40, paired t test comparison with time 0 min. (e and f) Live-cell microscopy of yeast cells expressing Heh1-GFP 120 min after the release from the G1 arrest in the minimal media without (e) or with (f) 2.5 µM myriocin. Scale bar, 2 µm. Mother cells show a shmoo from the G1 arrest and are labeled M. Daughter cells are labeled D. (g) Live-cell microscopy of yeast cells expressing Heh1-GFP 100 min after the release before anaphase from the G1 arrest in minimal media with 2.5 µM myriocin shows no defects. (h) Live-cell microscopy of yeast cells expressing Heh1-GFP 120 min after the release before anaphase from the G1 arrest in minimal media with 10 µM cerulenin. (i) Western blot analysis of mitotic Clb2 levels after release from G1 arrest in the presence of 10 µM cerulenin. The PSTAIR antibody was used as a loading control. Relative levels of Clb2 quantified with ImageJ are shown. HPLC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry. Source data are available for this figure: SourceData F4.

LCB synthesis takes place upon entry into the cell cycle. (a) DNA content of yeast cells (n = 30,000) after release from a pheromone-induced G1 arrest without or with myriocin (2.5 µM) at indicated time points. The asterisk denotes the beginning of the S phase. (b) Western blot analysis of mitotic Clb2 levels after release from G1 arrest. The PSTAIR antibody was used as a loading control. Relative levels of Clb2 quantified with ImageJ are shown under the western mages. The percentage of budded cells quantified by visual inspection of DIC images are shown below (n = 200). (c) HPLC-MS/MS analysis of LCB and ceramides in yeast cells harvested at indicated time points after release from a G1 arrest. Log2 ratios of the lipid levels relative to time 0 min are shown. Columns represent experiments, and rows represent lipid species. Cer, ceramide; dh, dihydro; DHS, dihydrosphingosine; PHS, phytosphingosine. (d) Plots of the total lipid classes as a function of time of the lipidome data presented in Fig. 2 c. *P < 1 e-40, paired t test comparison with time 0 min. (e and f) Live-cell microscopy of yeast cells expressing Heh1-GFP 120 min after the release from the G1 arrest in the minimal media without (e) or with (f) 2.5 µM myriocin. Scale bar, 2 µm. Mother cells show a shmoo from the G1 arrest and are labeled M. Daughter cells are labeled D. (g) Live-cell microscopy of yeast cells expressing Heh1-GFP 100 min after the release before anaphase from the G1 arrest in minimal media with 2.5 µM myriocin shows no defects. (h) Live-cell microscopy of yeast cells expressing Heh1-GFP 120 min after the release before anaphase from the G1 arrest in minimal media with 10 µM cerulenin. (i) Western blot analysis of mitotic Clb2 levels after release from G1 arrest in the presence of 10 µM cerulenin. The PSTAIR antibody was used as a loading control. Relative levels of Clb2 quantified with ImageJ are shown. HPLC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry. Source data are available for this figure: SourceData F4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal