Inhibition of transcription by dactinomycin reveals a new characteristic of immunogenic cell stress (2020)
Figure 3

Figure 3. DACT induces a split ER stress response in U2OS.

(A, B) Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured.

(C, D) U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified.

(E, F) U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity.

(G, H) U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular.

(I, J) U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified.

(K-N) U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h (M).

Data information: Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM (A, C, E, G, I). Scale bars represent 20 µm (A, C, E, G, I) or 10 µm (K, M). For all bar charts, the mean ± SEM of three to five independent experiments is shown. Each value was compared to the control and the p-value was calculated with a Student's t-test: * p<0.05, ** p<0.01, *** p<0.001 (B, D, F, H). For peIF2α quantification statistics were calculated using pairwise multiple comparison with a Benjamin-Hochberg correction. Stars indicate differences of each treatment compared to the control in the wild-type cells whereas hashes indicate differences comparing the kinases knock-out cells to the wild-type cells for the same treatment: **/## p<0.01, ***/### p<0.001 (J). For ex vivo data, results are shown as dot plots with mean ± SD , and p-values were calculated using Student's t-test: * p<0.05, ** p<0.01 (L, N).