Autophagy and mitophagy are triggered by CLPP inhibition with consequences on mitochondrial ATP production. Bioinformatic analyses of proteomic data from MM1.S cells in which CLPP activity was suppressed by an inducible shRNA (A) or inhibited by A2-32-01 (B), showing upregulated pathways and individual proteins of note. (C) Induction of autophagy by CLPP inhibition in MM1.S and H929 cells as measured by a dye-based assay. (D) Mitophagy induction was similarly examined using a dye-based assay in MM1.S and H929 cells. Western blotting for microtubule-associated protein 1A/1B light chain 3B-phosphatidylethanolamine conjugate (LC3-II) is shown after pharmacologic inhibition (E) or inducible suppression (F) of CLPP. For panels E-F, the fold change in LC3-II levels based on densitometry is provided below each lane after adjustment for loading of β-tubulin and in relation to the vehicle control, which was arbitrarily set at 1.0. Fold changes in red were found to be statistically significant based on t tests performed on 3 replicates. (G) The fold change in abundance of respiratory chain complex I and IV subunits in MM1.S cells with doxy-induced CLPP knockdown at days 3 and 7 is shown compared with cells with a control shRNA vector and doxy. The complex I and IV subunit genes that are indicated by the numbering on the abscissa are presented in supplemental Table 3. (H) Cellular ATP content decreased with inhibition (left) or inducible knockdown (right) of CLPP in MM1.S cells. (I) ATP production was also studied using the Seahorse XF Real-Time ATP Rate Assay, which revealed decreases especially in mitochondrial ATP production in MM1.S cells exposed to A2-32-01. GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score.

Autophagy and mitophagy are triggered by CLPP inhibition with consequences on mitochondrial ATP production. Bioinformatic analyses of proteomic data from MM1.S cells in which CLPP activity was suppressed by an inducible shRNA (A) or inhibited by A2-32-01 (B), showing upregulated pathways and individual proteins of note. (C) Induction of autophagy by CLPP inhibition in MM1.S and H929 cells as measured by a dye-based assay. (D) Mitophagy induction was similarly examined using a dye-based assay in MM1.S and H929 cells. Western blotting for microtubule-associated protein 1A/1B light chain 3B-phosphatidylethanolamine conjugate (LC3-II) is shown after pharmacologic inhibition (E) or inducible suppression (F) of CLPP. For panels E-F, the fold change in LC3-II levels based on densitometry is provided below each lane after adjustment for loading of β-tubulin and in relation to the vehicle control, which was arbitrarily set at 1.0. Fold changes in red were found to be statistically significant based on t tests performed on 3 replicates. (G) The fold change in abundance of respiratory chain complex I and IV subunits in MM1.S cells with doxy-induced CLPP knockdown at days 3 and 7 is shown compared with cells with a control shRNA vector and doxy. The complex I and IV subunit genes that are indicated by the numbering on the abscissa are presented in supplemental Table 3. (H) Cellular ATP content decreased with inhibition (left) or inducible knockdown (right) of CLPP in MM1.S cells. (I) ATP production was also studied using the Seahorse XF Real-Time ATP Rate Assay, which revealed decreases especially in mitochondrial ATP production in MM1.S cells exposed to A2-32-01. GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score.

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