Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei
Figure 4
Cell death and induction of SLS by ER stressors.
A. Recovery of cells after DTT treatment. Cells were treated with 4 mM DTT for 0, 30, 60, 120 and 240 minutes, washed twice in PBS and suspended in growth media to the same concentration. Cell growth was monitored for 4 days. Symbols indicate the duration of DTT treatment. B. Northern blot analysis of RNA from cells treated with ER stressors. RNA (10 µg) was prepared from procyclic (1–2) or bloodstream trypanosomes (3) treated with i. 4 mM DTT; ii. 20 mM 2-deoxy-D-glucose for 0, 60, 120 or 240 minutes or iii. 4 mM DTT for 0, 180, 240 minutes and was subjected to Northern analysis with radio-labeled anti-sense RNA probe. The srRNA-1 or srRNA-2 probes were used for loading control. C. Immunofluorescence monitoring the changes in amount and localization of tSNAP42 upon treatment with ER stressors. Cells were treated with i. 4 mM DTT; ii. 20 mM 2-DG for 3 hours. The cells were fixed with 4% (v/v) formaldehyde for 25 min, incubated with tSNAP42 antibodies and detected as described in Materials and Methods. The nucleus was stained with DAPI. DIC- differential interference contrast. The localization of tSNAP42 is indicated with arrows. Scale bars. 5 µm. Enlargement of the nuclear area is framed.