Figure 1.
Figure 1. PINK1–Parkin regulate misfolded protein clearance from mitochondria. (A) Tet ON: WT OTC or ΔOTC-expressing HeLa cells with or without YFP-Parkin expression were treated with DOX for 48 h or for 48 h with a 24- or 48-h washout of DOX and then processed for Western blot analysis. (B) Quantification of Western blots described in A expressed as the percentage of OTC levels relative to OTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 4. For left graphs, from left to right, *, P = 0.03; **, P = 0.008; ***, P = 0.0004; ***, P = 0.0001; for right graphs, ***, P = 5.8 × 10−5; ***, P = 0.0001; **, P = 0.007; **, P = 0.0011; ***, P = 6.7 × 10−9. Asterisks lacking a black underline represent significance values relative to OTC levels after 48 h DOX treatment (i.e., 100%). (C) Western blot of Tet ON: ΔOTC-expressing HeLa cells with YFP-Parkin expression with or without a PINK1 KO background and with or without PINK1-V5 expression were treated with DOX for 48 h or for 48 h with a 24 or 48 h washout of DOX. (D) Quantification of Western blots described in C and expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 4. From left to right, ***, P = 2.4 × 10−6; ***, P = 3 × 10−8; *, P = 0.045; **, P = 0.006. (E) Western blot of Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin with or without an ATG5 KO background treated with DOX for 48 h or with DOX for 48 h followed by a 24- or 48-h washout of DOX. (F) Quantification of Western blots described in E expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n = 3. From left to right, **, P = 0.003; ***, P = 0.0005; *, P = 0.03. (G) Tet-ON: ΔOTC-expressing HeLa cells without Parkin expression, with or without a PINK1 KO background, and with or without PINK1-V5 expression were treated with DOX for 48 h or for 48 h with a 48-h washout of DOX and with or without 100 nM bafilomycin and 20 µM QVD treatment and then processed for Western blot analysis. (H) Quantification of Western blots as described in G expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 3. From left to right, ***, P = 9.93 × 10−5; **, P = 0.002; **, P = 0.008; *, P = 0.036; ***, P = 0.0005. (I) Western blot of HeLa cells stably expressing Tet-ON: WT OTC and ΔOTC in the same cell with YFP-Parkin expression after treatment with DOX for 48 h or 48 h with a 24-h washout of DOX with or without 200 nM bafilomycin treatment and 20 µM QVD after washout. Error bars indicate SD.

PINK1–Parkin regulate misfolded protein clearance from mitochondria. (A) Tet ON: WT OTC or ΔOTC-expressing HeLa cells with or without YFP-Parkin expression were treated with DOX for 48 h or for 48 h with a 24- or 48-h washout of DOX and then processed for Western blot analysis. (B) Quantification of Western blots described in A expressed as the percentage of OTC levels relative to OTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 4. For left graphs, from left to right, *, P = 0.03; **, P = 0.008; ***, P = 0.0004; ***, P = 0.0001; for right graphs, ***, P = 5.8 × 10−5; ***, P = 0.0001; **, P = 0.007; **, P = 0.0011; ***, P = 6.7 × 10−9. Asterisks lacking a black underline represent significance values relative to OTC levels after 48 h DOX treatment (i.e., 100%). (C) Western blot of Tet ON: ΔOTC-expressing HeLa cells with YFP-Parkin expression with or without a PINK1 KO background and with or without PINK1-V5 expression were treated with DOX for 48 h or for 48 h with a 24 or 48 h washout of DOX. (D) Quantification of Western blots described in C and expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 4. From left to right, ***, P = 2.4 × 10−6; ***, P = 3 × 10−8; *, P = 0.045; **, P = 0.006. (E) Western blot of Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin with or without an ATG5 KO background treated with DOX for 48 h or with DOX for 48 h followed by a 24- or 48-h washout of DOX. (F) Quantification of Western blots described in E expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n = 3. From left to right, **, P = 0.003; ***, P = 0.0005; *, P = 0.03. (G) Tet-ON: ΔOTC-expressing HeLa cells without Parkin expression, with or without a PINK1 KO background, and with or without PINK1-V5 expression were treated with DOX for 48 h or for 48 h with a 48-h washout of DOX and with or without 100 nM bafilomycin and 20 µM QVD treatment and then processed for Western blot analysis. (H) Quantification of Western blots as described in G expressed as the percentage of ΔOTC levels relative to ΔOTC levels after 48 h DOX treatment normalized to Hsp90 levels. n ≥ 3. From left to right, ***, P = 9.93 × 10−5; **, P = 0.002; **, P = 0.008; *, P = 0.036; ***, P = 0.0005. (I) Western blot of HeLa cells stably expressing Tet-ON: WT OTC and ΔOTC in the same cell with YFP-Parkin expression after treatment with DOX for 48 h or 48 h with a 24-h washout of DOX with or without 200 nM bafilomycin treatment and 20 µM QVD after washout. Error bars indicate SD.

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