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. 2021 Mar 5;13(4):e12461. doi: 10.15252/emmm.202012461

Figure EV3. Combinatorial treatments induce the CLEAR network associated with a blockage in autophagy.

Figure EV3

  1. Relative mRNA expression levels of selected CLEAR network genes in HCT116 cells treated as indicated for 16 h (NEN (N) 1.2 µM, domperidone (Domp, D) 30 µM, imipramine (Imi), desipramine (Desi), amitriptyline (Ami), each 30 µM, clomipramine (Clomi) 20 µM).
  2. Immunoblot analysis of whole cell lysate (WL), cytoplasmic (Cyto) and nuclear (Nu) fractions of HCT116 cells using antibodies against the indicated proteins upon treatment of the cells as indicated for 16 h (Control (Ctrl), NEN (N) 1.2 µM, domperidone (D) 30 µM). GAPDH and acetyl‐histone H3 (AC‐H3) served as markers for cytoplasmic and nuclear fraction, respectively.
  3. Immunoblot analysis and quantification (mean ± SD) of LC3‐II/LC3‐I ratios and P62 in HCT116 cells. Cultured cells were treated with DMSO (Control), NEN or NEN and imipramine (Imi) as indicated (NEN, 1.2 µM; Imi, 30 µM) for 16 h. Protein band signal intensities were normalized to vinculin (loading control), and the ratios LC3‐II bands LC3‐I (N = 3) and p62 levels (N = 3) were analyzed by one‐way ANOVA with Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
  4. Immunoblot analysis and quantification (mean ± SD) of LC3‐II/LC3‐I ratios and P62 in HCT116 cells. Cultured cells were treated with DMSO (Control), NEN or NEN and amitriptyline (Ami) as indicated (NEN, 1.2 µM; Imi, 30 µM) for 16 h. Protein band signal intensities were normalized to vinculin (loading control), and the ratios LC3‐II bands LC3‐I (N = 3) were analyzed by one‐way ANOVA with Tukey post hoc test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Data information: In (A), data are presented as mean (SD) and were analyzed by a one‐way ANOVA with Tukey post hoc test. In (A), for the graphs with Imi, Ami, Desi and Clomi treatments (N = 5, besides NEN + Clomi (N = 4) and NEN + Desi (N = 4)), significance is indicated for the comparison of combinatorial treatments (NEN + individual TCAs) to controls. In (A), graphs with Domp (D) treatments (N = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P‐values for all comparisons are listed in Appendix Table S1.