STF-62247 and pimozide induce autophagy and autophagic cell death in mouse embryonic fibroblasts

Abstract

Induction of autophagy can have beneficial effects in several human diseases, e.g. cancer and neurodegenerative diseases (ND). Here, we therefore evaluated the potential of two novel autophagy-inducing compounds, i.e. STF-62247 and pimozide, to stimulate autophagy as well as autophagic cell death (ACD) using mouse embryonic fibroblasts (MEFs) as a cellular model. Importantly, both STF-62247 and pimozide triggered several hallmarks of autophagy in MEFs, i.e. enhanced levels of LC3B-II protein, its accumulation at distinct cytosolic sites and increase of the autophagic flux. Intriguingly, autophagy induction by STF-62247 and pimozide resulted in cell death that was significantly reduced in ATG5- or ATG7-deficient MEFs. Consistent with ACD induction, pharmacological inhibitors of apoptosis, necroptosis or ferroptosis failed to protect MEFs from STF-62247- or pimozide-triggered cell death. Interestingly, at subtoxic concentrations, pimozide stimulated fragmentation of the mitochondrial network, degradation of mitochondrial proteins (i.e. mitofusin-2 and cytochrome c oxidase IV (COXIV)) as well as a decrease of the mitochondrial mass, indicative of autophagic degradation of mitochondria by pimozide. In conclusion, this study provides novel insights into the induction of selective autophagy as well as ACD by STF-62247 and pimozide in MEFs.

Figure 1

STF-62247 and pimozide trigger autophagy in MEFs. (A,B) Lysates from untreated Atg5^+/+, Atg5^−/− (A) and Atg7^+/+, Atg7^−/− (B) MEFs were subjected to Western blotting with vinculin as loading control. The asterisk depicts free ATG5 which is present only in the absence of ATG7. (C) Atg5^+/+ and Atg5^−/− MEFs were treated with 20 µM STF-62247 or 15 µM pimozide while Atg7^+/+ and Atg7^−/− MEFs were treated with 40 µM STF-62247 or 10 µM pimozide for 24 hours followed by Western blotting with vinculin as loading control. The asterisk depicts free ATG5 which is present only in the absence of ATG7. The dotted line indicates representations of lysates from two independent experiments. (D,E) Atg5^+/+ cells were treated with 20 µM STF-62247 or 15 µM pimozide (D) and Atg7^+/+ cells were treated with 40 µM STF-62247 or 10 µM pimozide (E) for the indicated time points followed by Western blotting with vinculin as loading control. For quantification, LC3B-II protein levels of Atg5^+/+ and Atg7^+/+ MEFs were normalized to vinculin protein levels and expressed as fold changes compared to time point 0. p-values were calculated from 3-8 independent experiments. Uncropped blots are presented in Suppl. Fig. 3–7. STF = STF-62247, PIMO = pimozide, ns = not significant, nd = not determined.

Figure 2

STF-62247 and pimozide lead to a strong accumulation of endogenous LC3B-II protein in MEFs. (A,B) Atg5^+/+ and Atg5^−/− MEFs were treated with 20 µM STF-62247, 15 µM pimozide or 7.5 µM ABT-737/10 µM etoposide for 24 hours (A). Atg7^+/+ and Atg7^−/− MEFs were treated with 40 µM STF-62247, 10 µM pimozide or 7.5 µM ABT-737/10 µM etoposide for 24 hours (B). Formation of LC3B puncta was imaged using anti-LC3B immunofluorescence staining. Representative images over 25 sites per sample are shown. (C,D) Quantification of mean LC3B puncta per cell upon STF-62247, pimozide or ABT-737/etoposide treatment of Atg5^+/+ and Atg5^−/− (C) or Atg7^+/+ and Atg7^−/− (D) MEFs. Mean and SEM of three independent experiments performed for 25 sites per sample are shown. Significances after drug treatment of Atg5^+/+, Atg5^−/− and Atg7^+/+, Atg7^−/− cells are calculated versus untreated cells of the corresponding cell line. Scale bar = 30 µM. **p < 0.01, ***p < 0.001. ut = untreated, STF = STF-62247, PIMO = pimozide, eto = etoposide.

Figure 3

STF-62247 and pimozide lead to enhanced autophagic flux in MEFs. (A,C) Atg5^+/+ MEFs were treated for 4 hours with 20 µM STF-62247 (A) or 15 µM pimozide (C) in the absence or presence of 40 nM BafA1. Protein levels were detected by Western blotting with vinculin as loading control. (B,D) Atg7^+/+ MEFs were treated for 16 hours with 40 µM STF-62247 (B) or for 4 hours with 10 µM pimozide (D) in the absence or presence of 40 nM BafA1. Protein levels were detected by Western blotting with vinculin as loading control. For quantification, LC3B-II protein levels were normalized to vinculin protein levels and expressed as fold changes compared to the untreated sample. (E,F) Atg5^+/+ and Atg7^+/+ MEFs were transfected with mRFP-EGFP-LC3B followed by treatment with 20 µM STF-62247 or 15 µM pimozide and 40 µM STF-62247 or 10 µM pimozide in the absence or presence of 40 nM BafA1, respectively, for 8 hours (Atg5^+/+) or for 12 hours (Atg7^+/+). Images were acquired by confocal microscopy. Arrows highlight mRFP⁺ /EGFP^- puncta. (G,H) The numbers of mRFP⁺/EGFP⁻ and mRFP⁺/EGFP⁺ dots per cell were quantified after treatment of Atg5^+/+ (G) and Atg7^+/+ (H) MEFs with 20 µM STF-62247 or 15 µM pimozide and 40 µM STF-62247 or 10 µM pimozide, respectively, in the absence and presence of 40 nM BafA1. Mean and SEM of three independent experiments are shown. 21-51 cells were quantified per sample. Red stars indicate significances of mRFP⁺ /EGFP^- dots in treated versus untreated cells. Black stars indicate significances of the sum of mRFP⁺ /EGFP^- and mRFP⁺ /EGFP⁺ dots in cells treated with STF-62247 or PIMO in combination with BafA1 versus cells treated with BafA1 alone. Scale bar = 20 µM. Uncropped blots are presented in Suppl. Fig. 8. *p < 0.05, **p < 0.01, ***p < 0.001. ut = untreated, BafA1 = bafilomycin A1, STF = STF-62247, PIMO = pimozide.

Figure 4

STF-62247 and pimozide induce ATG5- and ATG7-dependent cell death in MEFs. (A–D) Atg5^+/+, Atg5^−/− (A) and Atg7^+/+, Atg7^−/− (B) MEFs were treated with the indicated concentrations of STF-62247 for 48 hours. Atg5^+/+, Atg5^−/− (C) and Atg7^+/+, Atg7^−/− (D) MEFs were treated with the indicated concentrations of pimozide for 48 hours. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Data are presented as mean and SEM of three to five independent experiments performed in triplicate. Significances were calculated against WT cells treated with the same drug concentration. *p < 0.5, **p < 0.01, ***p < 0.001. STF = STF-62247, PIMO = pimozide.

Figure 5

STF-62247 and pimozide induce autophagy-dependent cell death in a time-dependent manner in MEFs. (A–F) Atg5^+/+ and Atg5^−/− MEFs were treated for 24, 48 and 72 hours with 20 µM STF-62247 (A) or 15 µM pimozide (C) and for 8, 24 and 48 hours with 7.5 µM ABT-737/10 µM etoposide (E). Atg7^+/+ and Atg7^−/− MEFs were treated for 24, 48 and 72 hours with 40 µM STF-62247 (B) or 10 µM pimozide (D) and for 8, 24 and 48 hours with 7.5 µM ABT-737/10 µM etoposide (F). Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Data are presented as mean and SEM of three to five independent experiments performed in triplicate. Significances were calculated against WT cells treated with the same drug concentration. *p < 0.5, **p < 0.01, ***p < 0.001. STF = STF-62247, PIMO = pimozide.

Figure 6

STF-62247- and pimozide-induced cell death shows no major hallmarks of apoptosis, necroptosis or ferroptosis. (A,B,D,E,G,H) Atg5^+/+ MEFs were pre-treated for one hour with 20 µM zVAD.fmk (A), 20 µM Nec1s (D) or 5 µM Fer-1 (G) followed by treatment with 20 µM STF-62247 or 15 µM pimozide for 48 hours. Atg7^+/+ MEFs were pre-treated for one hour with 20 µM zVAD.fmk (B), 20 µM Nec1s (E) or 5 µM Fer-1 (H) followed by treatment with 40 µM STF-62247 or 10 µM pimozide for 48 hours. (C) Atg5^+/+ MEFs were pre-treated with 20 µM zVAD.fmk for one hour followed by treatment with 7.5 µM ABT-737/10 µM etoposide treatment for 48 hours. (F) HT-29 cells were pre-treated with 20 µM Nec1s for one hour followed by treatment with 1 ng/ml TNFα, 0.5 µM BV6 and 20 µM zVAD.fmk for 48 hours. (I) Atg5^+/+ MEFs were pre-treated with 5 µM Fer-1 for one hour followed by treatment with 3 µM Erastin for 48 hours. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Data are presented as mean and SEM of three independent experiments performed in triplicate. Significances were calculated against control cells treated with the same concentration of STF-62247 or pimozide. *p < 0.5, **p < 0.01, ***p < 0.001. Ctrl. = control, STF = STF-62247, PIMO = pimozide, eto = etoposide, TBZ = TNFα, BV6 and zVAD.fmk.

Figure 7

Pimozide-mediated induction of autophagy induces features of mitophagy in mCherry-Parkin-expressing MEFs. (A) Atg5^+/+ and Atg5^−/− MEFs were transfected with mCherry-Parkin and expression of mCherry-Parkin was verified by Western blotting with GAPDH as loading control. mCherry-Parkin (expected molecular weight = 81 kDa) is depicted by an arrow. (B) mCherry-Parkin-expressing Atg5^+/+ MEFs were treated with 10 µM FCCP or 10 µM pimozide for 8 hours followed by analysis of mitochondrial morphologies using anti-TOMM20 immunofluorescence staining. Arrows depict disruption of the mitochondrial network. (C) mCherry-Parkin-expressing Atg5^+/+ MEFs were treated with 10 µM FCCP or 10 µM pimozide for the indicated time points followed by Western blotting with GAPDH as loading control. (D) mCherry-Parkin-expressing Atg5^+/+ and Atg5^−/− MEFs were treated with 10 µM FCCP or 10 µM pimozide for 8 hours followed by q-PCR in order to assess the mitochondrial mass determined as the ratio between the DNA levels of the mitochondrial gene Cytochrome C oxidase and the nuclear gene β-actin. Scale bar = 30 µM. Data are presented as mean and SEM of three to four independent experiments performed in triplicate. Significances were calculated against untreated cells of the same cell line. Uncropped blots are presented in Suppl. Fig. 9–13. **p < 0.01. UT = untreated, FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, PIMO = pimozide.