Additional file 1 of The A’-helix of CYP11A1 remodels mitochondrial cristae
Creators: name: Rosal, Karen G.
name: Chen, Wei-Yi
name: Chung, Bon-chu
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Table S1. Key resources used in this study. Figure S1. Mass spectra analysis of stable clone C1 with (C1 +) and without CYP11A1 (C1-) after immunoprecipitation (IP). (A) Spectra from mass analysis of proteins eluted peaks corresponding to peptides. (B) Sequences of CYP11A1 and Hsp60. The polypeptides identified by MALDI-TOF are shown in red. Figure S2. The original uncropped data of Fig. 1A. Western blot showing stable clones C1 and C4 overexpressing CYP11A1 when induced with doxycycline (doxy). Boxes indicate bands identified by corresponding antibodies. β-actin was used as a loading control. Figure S3. The original uncropped data of Fig. 2A. Immunohistochemical images of zebrafish Cyp11a1-EGFP and Cyp11a2-EGFP transfected in COS1 cells showing localization of EGFP in mitochondria. TOM20 (red) is a marker for mitochondria, and DAPI (blue) stains the nucleus. Boxes mark the images shown in Fig. 2A. Figure S4. The original uncropped immunofluorescence data of Fig. 3. Immunohistochemistry images of CYP11A1-fragments-EGFP transfected in COS1 cells showing localization of EGFP in mitochondria. TOM20 (red) is a marker for mitochondria, and DAPI (blue) stains the nucleus. Boxes mark the images shown in Fig. 3. Figure S5. The original uncropped immunoblot data of Fig. 3B. Partitioning of EGFP-fused CYP11A1 fragments and anchoring region (A’) examined by immunoblotting after alkaline buffer extraction and ultracentrifugation. The EGFP and 39-EGFP (abbreviated as 39) proteins were in the supernatant, while the 85-, and 521-EGFP proteins were in the pellet. Asterisk indicates non-specific binding of antibody used (T: total; P: pellet; S: supernatant). TOM20 is a control for membrane protein, and cytochrome c (Cyt C) is a control for soluble protein. Boxes mark the bands shown in Fig. 3B. Figure S6. The original uncropped immunoblot data of Fig. 3D. Partitioning of EGFP-fused CYP11A1 fragments in a stable clone (SC) detected by Western blots. The EGFP protein fused to AA#1–39 plus the anchoring region (SC39 + A’) and AA#1–85 (SC85) also goes to the pellet. The asterisk indicates non-specific bands (T: total; P: pellet; S: supernatant). Boxes mark the bands shown in Fig. 3D. Figure S7. The original uncropped immunoblot data of Fig. 4A. Western blot showing similar amounts of components of the electron transport complex (CI-CV) from stable clones of CYP11A1 (C1, C4) with or without induction of CYP11A1. β-actin was used as loading control. Boxes indicate the bands detected by antibody used for each sample. The asterisk indicates non-specific bands. Figure S8. The original uncropped immunoblot data of Fig. 5A-E. Verification of CYP11A1 and Hsp60 interaction and the effect of Hsp60 depletion on CYP11A1. Boxes mark the bands shown in Fig. 5. Figure S9. The original uncropped immunoblot data of Fig. 6A and C. Inverse relationship of CYP11A1 and Mic10 complexes. Boxes mark the bands shown in Fig. 6. Figure S9. Inverse relationship of CYP11A1 and Mic10 complexes. (A) Western Blot showing MIC10 and SCC complexes after separation of protein complexes from mitochondria of stable clones that overexpress CYP11A1 by blue-native gel electrophoresis. Reduction of Mic10 complex is shown by decreased band intensity with induced CYP11A1 complex. (B) Western blot of proteins separated by SDS-PAGE showing unchanged MIC10 amount in stable clones in the presence ( +) or absence (−) of doxycycline (doxy) induction in stable clones (C1, C2, C4). Asterisk indicates non-specific bands. Figure S10. BN-PAGE of electron supercomplexes using stable clone C4. Electron supercomplexes were detected in COS1 and C4 with ( +) and without (-) doxycycline (doxy). Visible bands of electron supercomplexes are labeled with broken lines. Figure S11. Measurement of mitochondrial membrane potential in stable clones of CYP11A1. A. Fluorescent images of COS1, C1, and C4 cells after induction of protein overexpression by doxycycline (doxy). GFP (green) shows the presence of CYP11A1 after induction with doxycycline. TMRM staining (red) shows mitochondrial membrane potential. (B). TMRM intensity was quantified and compared.