| Antibodies |
|
|
|
| Actin |
Santa Cruz |
Cat#sc-47778; clone C4; RRID:AB_2714189 |
| Alexa Fluor goat anti-chicken IgY 488 nm |
Invitrogen |
Cat#A32931; RRID:AB_2762843 |
| Alexa Fluor goat anti-mouse IgG 405 nm |
Invitrogen |
Cat#A31553; RRID:AB_221604 |
| Alexa Fluor goat anti-mouse IgG 488 nm |
Invitrogen |
Cat#A32723; RRID:AB_2633275 |
| Alexa Fluor goat anti-mouse IgG 594 nm |
Invitrogen |
Cat#A32742; RRID:AB_2762825 |
| Alexa Fluor goat anti-mouse IgG 633 nm |
Invitrogen |
Cat#A21052; RRID:AB_2535719 |
| Alexa Fluor goat anti-rabbit IgG 405 nm |
Invitrogen |
Cat#A31556; RRID:AB_221605 |
| Alexa Fluor goat anti-rabbit IgG 488 nm |
Invitrogen |
Cat#A32731; RRID:AB_2633280 |
| Alexa Fluor goat anti-rabbit IgG 594 nm |
Invitrogen |
Cat#A32740; RRID:AB_2762824 |
| Alexa Fluor goat anti-rabbit IgG 633 nm |
Invitrogen |
Cat#A21071; RRID:AB_2535732 |
| Alpha-tubulin |
Sigma |
Cat#T9026; clone DM1A; RRID:AB_477593 |
| Desmoglein 1 |
Abcam |
Cat#Ab12077; Clone 27B2; RRID:AB_2093304 |
| Desmoglein 1 |
Santa Cruz |
Cat#sc-137164; Clone B11; RRID:AB_2093310 |
| DRP1 |
Abcam |
Cat#Ab56788; RRID:AB_941306 |
| DRP1 |
Santa Cruz |
Cat#sc-101270; Clone 6Z-82; RRID:AB_2093545 |
| DRP1 |
Sigma |
Cat#HPA039324; RRID:AB_2676443 |
| Filaggrin |
Abcam |
Cat#Ab3137; RRID:AB_303542 |
| FIS1 |
Santa Cruz |
Cat#sc-376447; Clone B-5; RRID:AB_11149382 |
| FIS1 |
Sigma |
Cat#HPA017430; RRID:AB_1848608 |
| GAPDH |
Abcam |
Cat#Ab9484; RRID:AB_307274 |
| GFP |
Aves |
Cat#GFP-1020; RRID:AB_10000240 |
| Halo tag |
Promega |
Cat#G9281; RRID:AB_713650 |
| Hsp60 |
Sigma |
Cat#HPA001523; RRID:AB_1079028 |
| Keratin 10 |
Abcam |
Cat#Ab76318; RRID:AB_1523465 |
| Keratin 14 |
Abcam |
Cat#Ab7800; RRID:AB_306091 |
| LC3b |
Abcam |
Cat#Ab48394; RRID:AB_881433 |
| Loricrin |
Abcam |
Cat#Ab24722; RRID:AB_448258 |
| NIX/BNIP3L |
Sigma |
Cat#HPA015652; RRID:AB_1845406 |
| NIX/BNIP3L |
Santa Cruz |
Cat#sc-166332; Clone H-8; RRID:AB_2066782 |
| NIX/BNIP3L |
Abcam |
Cat#Ab109414; RRID:AB_10863865 |
| SNAP tag |
New England BioLabs |
Cat#P9310S; RRID:AB_10631145 |
| TOM20 |
Santa Cruz |
Cat#sc-17764; Clone F-10; RRID:AB_628381 |
| TOM20 |
Abcam |
Cat#Ab56783; RRID:AB_945896 |
| TOM20 |
Sigma |
Cat#HPA011562; RRID:AB_1080326 |
|
| Bacterial and virus strains |
|
|
|
| Competent E. coli
|
Thermo-Fisher |
#C737303 |
|
| Biological samples |
|
|
|
| Human skin tissue |
Univ. of Pennsylvania Skin Biology and Disease Resource-based Center |
https://dermatology.upenn.edu/sbdrc/ |
| Normal human epidermal keratinocytes |
Univ. of Pennsylvania Skin Biology and Disease Resource-based Center |
https://dermatology.upenn.edu/sbdrc/ |
|
| Chemicals, peptides, and recombinant proteins |
|
|
|
| 3,3′,5-tri-iodo-L-thyronine |
Sigma |
Cat#T6397 |
| Adenine |
Sigma |
Cat#A2786 |
| Apo-transferrin |
Sigma |
Cat# T1147 |
| Bafilomycin A1 (BafA1) |
Sigma |
Cat#B1793 |
| Bovine serum albumin (BSA) |
Sigma |
Cat#A9647 |
| Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) |
Sigma |
Cat#C2759 |
| Cholera toxin |
Sigma |
Cat#C8052 |
| Cobalt chloride (CoCl2) |
Sigma |
Cat#15862 |
| Collagen type 1, high-concentration rat tail |
Corning |
Cat#354249 |
| Deferiprone (DFP) |
Sigma |
Cat#Y0001976 |
| Dimethyloxalylglycine (DMOG) |
Sigma |
Cat#D3695 |
| Dimethyl sulfoxide (DMSO) |
Sigma |
Cat#D8418 |
| Dispase II |
StemCell Technologies |
Cat#07913 |
| DMEM |
Corning |
Cat#10–017-CM |
| DMEM 10X |
Sigma |
Cat#D2554 |
| Fetal bovine serum (FBS) |
HyClone |
Cat#SH3007103 |
| JF646-Halo Ligand |
Lavis Lab, HHMI Janelia Research Campus, Ashburn, VA, USA |
https://www.janelia.org/open-science/janelia-fluor-dyes |
| EMbed-812 |
Electron Microscopy Sciences |
Cat#14120 |
| Ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC) |
AOBIOUS |
Cat#AOB13386
|
| EUK134 |
Sigma |
Cat#SML0743 |
| Formalin, 10%, neutral buffered |
Fisher |
Cat#SF100–4 |
| FuGENE6 transfection reagent |
Promega |
Cat#E269A |
| Gentamicin/Amphotericin |
GIBCO |
Cat#R01510
|
| GlutaMAX |
GIBCO |
Cat#35050061 |
| Ham’s F12 medium |
Corning |
Cat#10–080-CV |
| Hoechst 33342 |
Invitrogen |
Cat#H21492
|
| Human epidermal growth factor (EGF) |
Sigma |
Cat#E9644 |
| Human insulin |
Sigma |
Cat#91077C |
| Human keratinocyte growth supplement |
GIBCO |
Cat#S0015 |
| Hydrocortisone |
Sigma |
Cat#H0888 |
| Keratinocyte medium M154 |
GIBCO |
Cat#M154CFPRF500 |
| Keratinocyte serum-free medium kit (including bovine pituitary extract; epidermal growth factor; calcium chloride) |
GIBCO |
Cat#17005042 |
| Mdivi-1 |
Cell Signaling |
Cat#CAS-338967–87-6 |
| MG132 |
Sigma |
Cat#M8699) |
| MitoTracker Deep Red |
Invitrogen |
Cat#M22426
|
| Normal goat serum (NGS) |
Sigma |
Cat#G9023 |
| Opti-MEM |
GIBCO |
Cat#31985–070 |
| Penicillin/streptomycin |
Sigma |
Cat#P0781 |
| Polybrene |
Sigma |
Cat#H9268 |
| Prolong Gold mounting medium |
Invitrogen |
Cat#P36930
|
| Puromycin |
Sigma |
Cat# P8833 |
| SNAP-Cell 647-SIR |
New England BioLabs |
Cat#S9102S |
| Tetramethylrhodamine Ethyl Ester Perchlorate (TMRE) |
Invitrogen |
Cat#T669 |
| TrueBlack WB blocking buffer kit |
Biotium |
Cat#23013 |
| Trypsin-EDTA |
GIBCO |
Cat#25300054 |
| Xylenes |
Fisher |
Cat#X3F |
|
| Critical commercial assays |
|
|
|
| NucleoSpin Gel and PCR Clean-Up kit |
Takara |
Cat#740609 |
| Plasmid DNA Mini-prep Kit |
QIAGEN |
Cat#27106 |
| TransfeX Transfection Kit |
ATCC |
Cat#ACS4005 |
|
| Experimental models: cell lines |
|
|
|
| J2 3T3 murine fibroblasts |
James Rheinwald Lab, Harvard Univ., Cambridge, MA, USA |
N/A |
| hTERT-immortalized human epidermal keratinocytes (THEK) |
James Rheinwald Lab, Harvard Univ., Cambridge, MA, USA |
N/A |
| Phoenix HEK293T Phoenix viral packaging cells |
Garry Nolan Lab, Stanford Univ., Palo Alto, CA, USA |
N/A |
| THEK CRISPR Control KO line PG5 |
This paper |
N/A |
| THEK CRISPR NIX KO line 1 |
This paper |
N/A |
| THEK CRISPR NIX KO line 2 |
This paper |
N/A |
|
| Oligonucleotides |
|
|
|
| sgRNA control forward oligo: PG5SG1F |
This paper |
5′-CACCGGTATTCCGTGGGTGAACGGG-3′ |
| sgRNA control reverse oligo: PG5SG1R |
This paper |
5′-AAACCCCGTTCACCCACGGAATACC-3′. |
| sgRNA for NIX/BNIP3L forward oligo: BNIP3LSGRNAF1 |
This paper |
5′-CACCGGACATTGTCGGACAGCTGGC-3′ |
| sgRNA for NIX/BNIP3L reverse oligo: BNIP3LSGRNAR1 |
This paper |
5′-AAACGCCAGCTGTCCGACAATGTCC-3′ |
| PCR forward primer for control KO sequencing: PG5PCRF2 |
This paper |
5′-CCTCGGAAAACTCTCCTTCC-3′ |
| PCR reverse primary for control KO sequencing: PG5PCRR2 |
This paper |
5′-TGGATTGCTGTCCCTCTAGC-3′ |
| PCR forward primer for NIX/BNIP3L KO sequencing: BNIP3LPCRF1 |
This paper |
5′- CGGACTCGGCTTGTTGTGTT-3′ |
| PCR reverse primary for NIX/BNIP3L KO sequencing: BNIP3LPCRR1 |
This paper |
5′-GGGGCAGAGACTGCTCATTTT-3′ |
|
| Recombinant DNA |
|
|
|
| pcDNA3-SNAP-DRP1-K38A |
Smirnova et al. 1998 |
Addgene plasmid #45161 |
| pcDNA3.1-GFP-BNIP3L |
Fei et al. 2004 |
Addgene plasmid #17467 |
| pCLBW-Cox8-GFP-mCh |
Rojansky et al. 2016 |
Addgene plasmid #78520 |
| pdsRed2-Mito |
Thomas Schwarz Lab, Harvard Univ., Boston, MA, USA |
N/A |
| pLZRS |
Kathleen Green Lab, Northwestern Univ., Chicago, IL, USA |
N/A |
| pLZRS-beta-tubulin-mCh |
Kathleen Green Lab, Northwestern Univ., Chicago, IL, USA |
N/A |
| pLZRS-dsRed-Mito |
This paper |
N/A |
| pLZRS-GFP-Mito |
This paper |
N/A |
| pLZRS-GFP-NIX |
This paper |
N/A |
| pLZRS-GFP-NIX-2GA |
This paper |
N/A |
| pLZRS-GFP-NIX-WA |
This paper |
N/A |
| pLZRS-Halo-FIS1 |
This paper |
N/A |
| pLZRS-Mito-SNAP |
This paper |
N/A |
| pLZRS-SNAP-DRP1-K38A |
This paper |
N/A |
| pLZRS-TOM20-mCh |
This paper |
N/A |
| pmCherry-TOM20 |
Michael Davidson Lab, Florida State Univ., Tallahassee, FL, USA |
Addgene plasmid #55146 |
| pSEMS-Halo7Tag-hFIS1 |
Appelhans et al. 2012 |
Addgene plasmid #111136 |
| pSpCas9 (BB)-2A-GFP (PX458) |
Ran et al. 2013 |
Addgene plasmid #48138 |
|
| Software and algorithms |
|
|
|
| CRISPR/Cas9 Guide RNA Design |
GPP Web Portal, Broad Institute, Cambridge, MA, USA |
https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design |
| Fiji Image Anlysis |
ImageJ |
https://imagej.net/Fiji |
| Prism version 8.1.1 |
GraphPad |
N/A |
|
| Other |
|
|
|
| Deep 6-well culture plates |
Falcon |
Cat#355467 |
| Glass coverslips |
Fisher |
Cat#12–543A |
| Glass-bottom tissue culture dishes |
MatTek |
Cat#P35G-1.5–20-C |
| Transwell inserts |
Falcon |
Cat#353091 |
