Fig. 3: The blockage of autophagy in Csg2 deleted cells was related with the accumulation of ER calcium.
From: The ER calcium channel Csg2 integrates sphingolipid metabolism with autophagy
a Degradation of autophagic substrate GFP-Atg8 in WT cells, csg2Δ cells, and atg1Δ cells after starvation in SD-N medium with or without CaCl2 (100 mM), EDTA (5 mM), or EGTA (20 mM) was detected at the indicated times. The blots were probed with anti-GFP antibody and Pgk1 was used as a loading control. The samples derive from the same experiment and gels were processed in parallel. b WT cells, csg2Δ cells, and atg1Δ cells were checked for cell viability before and after starvation in SD-N medium with or without EDTA (5 mM) and EGTA (20 mM). The experiment was repeated independently for three times with similar results. c Vacuole transfer of autophagic marker GFP-Atg8 in csg2Δ cells was restored by addition of ion chelating agents EDTA and EGTA. Autophagic marker GFP-Atg8 was investigated by fluorescence assays in WT cells, csg2Δ cells, and atg1Δ cells after starvation in SD-N medium with or without EDTA (5 mM) or EGTA (20 mM). The experiment was repeated independently for three times and representative images were shown. Scale bars: 5 μm. d Vacuole transfer of autophagic substrate RFP-Ape1 in csg2Δ cells was restored by addition of ion chelating agents EDTA and EGTA. Transportation of the autophagic substrate RFP-Ape1 was investigated by fluorescence assays in WT cells, csg2Δ cells, and atg1Δ cells after starvation in SD-N medium with or without EDTA (5 mM) or EGTA (20 mM). The experiment was repeated independently for three times and representative images were shown. Scale bars: 5 μm.
